Cultivation, Collection and Processing of Herbal Drugs

 Chapter 6 

Cultivation, Collection and Processing of Herbal Drugs

INTRODUCTION

The crude drugs which reach the market and pharmaceutical industries will have passed through different stages that have some effect in the nature and amount of active constituents responsible for therapeutic activity. Those stages are to be concerned more in order to make a drug useful to the mankind by all means. This chapter concerns regarding such parameters which has some effect over plants.

Cultivation produces improved quality of plants. It helps in selecting the species, varieties or hybrids that have the desired phytoconstituents due to the controlled environmental growth better plant product is obtained and makes the collection and processing steps easier when compared to wild sources. Cultivation results in obtaining plants with maximum secondary metabolites. It leads to industrialization in the country by the regular supply of plants. Serves as a useful tool for research purposes.

The advantages of cultivation may be briefly summarized as follows:

• It ensures quality and purity of medicinal plants. Crude drugs derive theirutility from chemical contents in them. If uniformity is maintained in all operations during the process of cultivation, drugs of highest quality can be obtained. Cultivation of rhizomes demands an adequate quantity of fertilizers and proper irrigation. Systematic cultivation results in raising a crop with maximum content of volatile oil and other constituents. The examples of ginger, turmeric and liquor ice can be cited to illustrate this point. If the cultivated plants are kept free of weeds, the contamination of crude drugs can be conveniently avoided.

• Collection of crude drugs from cultivated plants gives a better yield and therapeutic quality. However, it is a skilled operation and requires some professional excellence, if the collection of crude drugs for market is done from cultivated plants by skilled and well experienced personnel, the high yield and therapeutic quality of drugs can be maintained. For example, collection of latex from poppy capsules and oleoresins from Pinus species, if done by experienced persons, can result in better yield of crude drugs. Preservation of green colour of senna leaves and minimizing the deterioration of cardiac glycosides in freshly collected leaves of digitalis can be achieved only by highly skilled labor.

• Cultivation ensures regular supply of a crude drug. In other words, cultivation is a method of crop-planning. Planning a crop cultivation regularizes its supply and as a result the industries depending upon crude drugs do not face problem of shortage of raw material.

• The cultivation of medicinal and aromatic plants also leads to industrialization to a greater extent. The cultivation of coffee and cocoa in Kerala has given rise to several cottage and small-scale industries. The cultivation of cinchona in West Bengal has led to the establishment of the cinchona-alkaloid factory near Darjeeling. The government owned opium factory at Ghaziabad is an eloquent testimony to the significance of well planned cultivation of poppy.

• Cultivation permits application of modern technological aspects such as mutation, polyploidy and hybridization.

 SOILS, SEEDS AND PROPAGATION MATERIAL

• The physical, chemical and microbiological properties of the soil play a crucial role in the growth of plants. Water holding capacity of different sizes of soil too affects the plants. The calcium present in the soil would be very much useful for some plants where as the others does not require calcium. The seed to be used for cultivation should be identified botanically, showing the details of its species, chemotype and origin. The seeds should be 100% traceable. The parent material should meet standard requirements regarding thepurity and germination. It should be free from pests and diseases in order to guarantee healthy plant growth. Preference should be given to the resistant or tolerant species. Plant materials or seeds derived from genetically modified organisms have to comply with national and European Union regulations. Season when the seeds should be sown and at what stage a seed should be sown should be predetermined. Few seeds such as cinnamon losses its viability if stored for long period and the percentage of germination would be less for the seeds which were long stored.

Methods of Plant Propagation

• Medicinal plants can be propagated by two usual methods as applicable to nonmedicinal plants or crops. These methods are referred as sexual method and asexual method. Each of these methods has certain advantages, and also, disadvantages.

 Sexual method (seed propagation)

In case of sexual method, the plants are raised from seeds and such plants are known as seedlings. The sexual method of propagation enjoys following advantages:

• Seedlings are long-lived (in case of perennial drugs) and bear more heavily (in case of fruits). Plants are sturdier.

• Seedlings are comparatively cheaper and easy to raise.

• Propagation from seed has been responsible for production of some chance-seedlings of highly superior merits which may be of great importance to specific products, such as orange, papaya, etc.

•  In case of plants where other vegetative methods cannot be utilized, propagation from seeds is the only method of choice.

Sexual method suffers from following limitations

• Generally, seedling trees are not uniform in their growth and yielding capacity, as compared to grafted trees.

• They require more time to bear, as compared to grafted plants.

• The cost of harvesting, spraying of pesticides, etc. is more as compared to grafted trees.

• It is not possible to avail of modifying influence of root stocks on scion, as in case of vegetatively propagated trees.

For propagation purpose, the seeds must be of good quality. They should be capable a high germination rate, free from diseases and insects and also free from other seeds, used seeds and extraneous material. The germination capacity of seeds is tested by rolled towel test, excised embryo test, etc. The seeds are preconditioned with the help of scarcification to make them permeable to water and gases, if the seeds are not to be germinated in nearfuture, they should be stored in cool and dry place to maintain their germinating power. Long storage of seeds should be avoided.

Before germination, sometimes a chemical treatment is given with stimulants like gibberellins, cytokinins, ethylene, thiourea, potassium nitrate or sodium hypochlorite. Gibbereilic acid (GA3 ) promotes germination of some type of dormant seeds and stimulates the seedling growth. Many freshly harvested dormant seeds germinate better after soaking in potassium nitrate solution. Thiourea is used for those seeds which do not germinate in dark or at high temperatures.

Methods of sowing the seeds

Numerous methods of sowing the seeds of the medicinal plants are in practice. Few of them using seeds for cultivation are described

• Broadcasting: If the seeds are extremely small the sowing is done by broadcasting method. In this method the seeds are scattered freely in well prepared soil for cultivation. The seeds only need raking. If they are deeply sown or covered by soil, they may not get germinated. Necessary thinning of the seedlings is done by keeping a specific distance, e.g. Isabgol, Linseed, Sesame, etc.

• Dibbling: When the seeds of average size and weight are available, they are sown byplacing in holes. Number of seeds to be put in holes vary from three to five, depending upon the vitality, sex of the plants needed for the purpose and the size of the plant coming out of the seeds.

• For example, in case of fennel four to five fruits are put in a single hole keeping suitable distance in between two holes. In case of castor, only two to three seeds are put. In case of papaya, the plants are unisexual and only female plants are desired for medicinal purposes. Hence, five to six seeds are put together and after the sex of the plants is confirmed, healthy female plant is allowed to grow while male plants and others are removed.

• Miscellaneous: Many a times the seeds are sown in nursery beds. The seedlings thus produced are transplanted to farms for further growth, such as cinchona, cardamom, clove, digitalis, capsicum, etc.

• Special treatment to seeds: To enhance germination, special treatments to seeds may be given, such as soaking the seeds in water for a day e.g. castor seeds and other slow-germinating seeds. Sometimes, seeds are soaked in sulphuric acid e.g. henbane seeds. Alter natively, testa is partially removed by grindstone or by pounding seeds with coarse sand, e.g. Indian senna. Several plant hormones like gibberellins, auxins are also used.

 Asexual method

In case of asexual method of vegetative propagation, the vegetative part of a plant, such as stem or root, is placed in such an environment that it develops into a new plant.

Asexual propagation enjoys following advantages:

• There is no variation between the plant grown and plant from which it is grown. As such, the plants are uniform in growth and yielding capacity. In case of fruit trees, uniformity in fruit quality makes harvesting and marketing easy.

•  Seedless varieties of fruits can only be propagated vegetatively e.g. grapes, pomegranates and lemon.

• Plants start bearing earlier as compared to seedling trees.

• Budding or grafting encourages disease-resistant varieties of plants

• Modifying influence of rootstocks on scion can be availed of.

•  Inferior or unsuitable varieties can be over-looked.

It suffers from following disadvantages:

• In comparison to seedling trees, these are not vigorous in growth and are notlong-lived.

•  No new varieties can be evolved by this method.

Asexual method of vegetative propa gation consists of three types:

• Natural methods of vegetative propagation.

• Artificial methods of vegetative propagation. 

• Aseptic method of micropropagation (tissue-culture).

Natural methods of vegetative propagation: It is done by sowing various parts of the plants in well prepared soil. The following are the examples of vegetative propagation.

Artificial methods of vegetative propagations: The method by which plantlets or seedlings are produced from vegetative part of the plant by using some technique or process is known as artificial method of vegetative propagation. These methods are classified as under:

Cuttings

• Stem cuttings

• Soft wood cuttings: Barberry.

• Semi hard wood cuttings: Citrus, camellia.

• Root cuttings: Brahmi.

• Leaf cuttings: Podophyllum.

• Leaf bud cuttings

• Hard wood cuttings: Orange, rose and bougainvillea

Layering 

• Simple layering: Guava, lemon

• Serpentine layering: jasmine, clematis

• Air layering (Gootee): Ficus, mango, bougainvillea, cashew nut

• Mount layering

• Trench layering

Grafting

• Whip grafting: Apple and rose

• Tongue grafting

• Side grafting: Sapota and cashew nut

• Approach grafting: Guava and Sapota

• Stone grafting: Mango

• Tip layering

Aseptic methods of micropropagation (tissue culture)

 It is a novel method for propagation of medicinal plants. In micropropagation, the plants are developed in an artificial medium under aseptic conditions from fine pieces of plants like single cells, callus, seeds, embryos, root tips, shoot tips, pollen grains, etc. They are also provided with nutritional and hormonal requirements.

Preparation and Types of Nursery Beds

For various genuine reasons, seeds cannot be sown directly into soil i.e. very small size (Isabgol, tulsi) high cost, poor germination rate and long germination time (Cardamom, Coriander). Under such circumstances, seeds are grown into the nursery bed which not only is economical, but one can look after the diseases (if any) during germination period. Small size of beds can be irrigated conveniently along with fertilizers, as and when necessary. There are four types of nursery beds.

• Flatbed method

• Raised bed method

• Ridges and furrow method

• Ring and basin method

Taking into consideration the amount of water and type of soil required for a particular seed one should select the type.

Methods of Irrigation

Water is essential for any type of cultivation. After studying the availably and requirement of water for a specific crop, one has to design his own irrigation system at the reasonable cost.

Following methods of irrigation are known traditionally in India. The cultivation has an option after giving due consideration to the merits and demerits of each.

• Hand watering: economical and easy to operate.

• Flood watering: easy to operate, results in wastage of water.

• Boom watering: easy to operate, but restricted utility.

• Drip irrigation: Scientific, systematic and easy to operate; costly.

• Sprinkler irrigation: Costly, gives good results.

GOOD AGRICULTURAL PRACTICES

• Depending on the method of cultivation different Standard Operating Procedures for cultivation should be followed by the cultivators. A suitable area for the cultivation and the standard operation procedures for the cultivation should be developed depending upon the needs of the plants. Medicinal and aromatic plants should not be grown in soils which are contaminated by sludge and not contaminated by heavy metals, residues of plant protection products and any other unnatural chemicals, so the chemical products (pesticide and herbicide) used should be with as minimum negative effect as possible, human faeces should be avoided. Depending upon the soil fertility and the nutritional requirement of medicinal plants the type of the fertilizer and the amount of the fertilizer to be used is determined. Products for chemical plant protection have to conform to the European Union’s maximum residue limits. Proper irrigation and drainage should be earned out according to the climatic condition and soil moisture. The soil used for cultivation should be well aerated. The use of pesticides and herbicides has to be documented. Irrigation should be minimized as much as possible and only be applied according to the needs of the plant. Water used for irrigation should be free from all possible forms of contaminants and should comply with national and European Union quality standards. The area for cultivation should be strictly prohibited from the contaminations like house garbage, industrial waste, hospital refuse and feces. Field management should be strengthened and proper measures like pruning, shading etc. should be provided for increasing the yield of the active constituent and maintain the consistency of the yield. The pests used should give high efficacy, hypotoxicity, and low residue at the minimum effective input so that the residue of pesticides are also reduced and protected from ecological environment.

• Application and storage of plant protection products have to be in conformity with the regulations of manufacturers and the respective national authorities. The application should only be earned out by qualified staff using approved equipment. The nutrient supply and chemical plant protection should secure the marketability of the product. The buyer of the batch should be informed about the brand, quantity and date of pesticide use in written.

• Though several countries in the world have a rich heritage of herbal drugs, very few have their claim for their procurement of crude drugs only from cultivated species. Our reliance on wild sources of crude drugs and the lack of information on the sound cultivation and maintaining technology of crude drugs have resulted in gradual depletion of raw material from wild sources. Though the cultivation of medicinal plants offers wide range of advantages over the wild sources, it can be an uneconomical process for some crude drugs which occur abundantly in nature e.g., nux vomica, acacia etc. On the other hand, crude drugs like cardamom, clove, poppy, tea, cinchona, ginger, linseed, isabgol, saffron, peppermint, fennel, etc. are obtained majorly from cultivated plants. The cultivation of crude drugs involves keen knowledge of various factors from agricultural and pharmaceutical sphere, such as soil, climate, rainfall, irrigation, altitude, temperature, use of fertilizers and pesticides, genetic manipulation and biochemical aspects of natural drugs. When all such factors are precisely applied, the new approach to scientific cultivation technology emerges out.

FACTORS AFFECTING CULTIVATION

• Cultivation of medicinal plants offers wide range of advantages over the plants obtained from wild sources. There are few factors to concern which have a real effect on plant growth and development, nature and quantity of secondary metabolites. The factors affecting cultivation are altitude, temperature, rainfall, length of day, day light, soil and soil fertility, fertilizers and pests. The effects of these factors have been studied by growing particular plants in different environmental conditions and observing variations. For example, a plant which is subjected to a particular environment may develop as a small plant which, when analysed shows high proportion of metabolite than the plants attained the required growth. Nutrients have the ability to enhance the production of secondary metabolites, at the same time they may reduce the metabolites as well.

Altitude

• Altitude is a very important factor in cultivation of medicinal plants. Tea, cinchona and eucalyptus are cultivated favorably at an altitude of 1,000–2,000 metres. Cinnamon and cardamom are grown at a height of 500–1000 metres, while senna can be cultivated at sea level. The following are the examples of medicinal and aromatic plants indicating the altitude for their successful cultivation (Table 6.1).

Altitude for Drug cultivation

Temperature

• Temperature is a crucial factor controlling the growth, metabolism and there by the yield of secondary metabolites of plants. Even though each species has become adapted to its own natural environment, they are able to exist in a considerable range of temperature. Many plants will grow better in temperate regions during summer, but they lack in resistance to withstand frost in winter.

Optimum Temperature for Drug Cultivation

Rainfall

• For the proper development of plant, rainfall is required in proper measurements. Xerophytic plants like aloes do not require irrigation or rainfall. The effects of rainfall on plants must be considered in relation to the annual rainfall throughout the year with the water holding properties of the soil. Variable results have been reported for the production of constituents under different conditions of rainfall. Excessive rainfall could cause a reduction in the secondary metabolites due to leaching of water soluble substances from the plants.

Day Length and Day Light

• It has been proved that even the length of the day has an effect over the metabolites production. The plants that are kept in long day conditions may contain more or less amount of constituents when compared to the plants kept in short day. For example peppermint has produced menthone, menthol and traces of menthofuran in long day conditions and only menthofuran in short day condition.

• The developments of plants very much in both the amount and intensity of the light they require. The wild grown plants would meet the required conditions and so they grow but during cultivation we have to fulfill the requirements of plants. The day light was found to increase the amount of alkaloids in belladonna, stramonium, cinchona, etc. Even the type of radiation too has an effect over the development and metabolites of plants.

Soil

Each and every plant species have its own soil and nutritive requirements. The three important basic characteristics of soils are their physical, chemical and microbiological properties. Soil provides mechanical support, water and essential foods for the development of plants. Soil consists of air, water, mineral matters and organic matters. Variations in particle size result in different soils ranging from clay, sand and gravel. Particle size influences the water holding capacity of soil. The type and amount of minerals plays a vital role in plant cultivation. Calcium favours the growth of certain plants whereas with some plants it does not produce any effects. The plants are able to determine their own soil pH range for their growth; microbes should be taken in to consideration which grows well in certain pH. Nitrogen containing soil has a great momentum in raising the production of alkaloids in some plants.

Depending upon the size of the mineral matter, the following names are given to the soil (Table 6.3).

Type of soil on the basis of particle size.

Depending upon the percentage covered by clay, soils are classified as under

Type of soil on the basis of percentage covered by clay.

Soil Fertility

• It is the capacity of soil to provide nutrients in adequate amounts and in balanced proportion to plants. If croppingis done without fortification of soil with plant nutrients, soil fertility gets lost. It is also diminished through leaching and erosion. Soil fertility can be maintained by addition of animal manures, nitrogen-fixing bacteria or by application of chemical fertilizers. The latter is time saving and surest of all above techniques.

Fertilizers and Manures

• Plant also needs food for their growth and development. What plants need basically for their growth are the carbon dioxide, sun-rays, water and mineral matter from the soil. Thus, it is seen that with limited number of chemical elements, plants build up fruits, grains, fibres, etc. and synthesize fixed and volatile oils, glycosides, alkaloids, sugar and many more chemicals.

Chemical fertilizers

• Animals are in need of vitamins, plants are in need of sixteen nutrient elements for synthesizing various compounds. Some of them are known as primary nutrients like nitrogen, phosphorus and potassium. Magnesium, calcium and sulphur are required in small quantities and hence, they are known as secondary nutrients. Trace elements like copper, manganese, iron, boron, molybdenum, zinc are also necessary for plant growths are known as micronutrients. Carbon, hydrogen, oxygen and chlorine are provided from water and air. Every element has to perform some specific function in growth and development of plants. Its deficiency is also characterized by certain symptoms.

Manures 

• Farmyard manure (FYM/compost), castor seed cake, poultry manures, neem and karanj seed cakes vermin compost, etc. are manures. Oil-cake and compost normally consists of 3–6% of nitrogen, 2% phosphates and 1–1.5% potash. They are made easily available to plants. Bone meal, fish meal, biogas slurry, blood meal and press mud are the other forms of organic fertilizers.

Biofertilizers

• Inadequate supply high costs and undesirable effects if used successively are the demerits of fertilizers or manures and hence the cultivator has to opt for some other type of fertilizer. Biofertilizers are the most suitable forms that can be tried. These consist of different types of micro organisms or lower organisms which fix the atmospheric nitrogen in soil and plant can use them for their day to day use. Thus they are symbiotic. Rhizobium, Azotobactor, Azosperillium, Bijericcia, Blue-green algae, Azolla, etc. are the examples of biofertilizers.

Pests and Pests Control

• Pests are undesired plant or animal species that causes a great damage to the plants. There are different types of pests; they are microbes, insects, non-insect pests and weeds.

Microbes

They include fungi, bacteria and viruses. Armillaria Root Rot (Oak Root Fungus) is a disease caused by fungi Armillaria mellea (Marasmiaceae) and in this the infected plant become nonproductive and very frequently dies within two to four years. Plants develop weak, shorter shoots as they are infected by the pathogen. Dark, root-like structures (rhizomorphs), grow into the soil after symptoms develop on plants. The fungus is favoured by soil that is continually damp. Powdery mildew is another disease caused by fungus Uncinula necato on leaves, where chlorotic spots appear on the upper surface of leaf. On fruit the pathogen appears as white, powdery masses that may colonize the entire berry surface. Summer Bunch Rot is a disease in which masses of black, brown, or green spores develop on the surface of infected berries caused by a variety microbes like Aspergillus niger, Alternaria tennis, Botrytis cinerea, Cladosporium herbarum, Rhizopus arrhizus, Penicillium sp., and others.

Fomitopsis pinicola (Sw.) P. Karst. Belonging to family Fomitopsidaceae causes a diseases known as red-belted fungus. Several other fungi attacks the medicinal plants, like Pythium pinosurn causes pythium rhizome rot, Septoria digitalis causing leaf spot, little leaf disease by Phywphthora cinnamomi Rands (Pythiaceae), etc.

Crown gall disease caused by Agrobacterium tumefaciens (Rblzobiaceae). Galls may be produced on canes, trunks, roots, and cordons and may grow to several inches in diameter. Internally galls are soft and have the appearance of disorganized tissue. The pathogen can be transmitted by any agent that contacts the contaminated material. Galls commonly develop where plants have been injured during cultivation or pruning. Xylella fastidiosa is a bacterium causes Pierce’s Disease, in this leaves become slightly yellow or red along margins and eventually leaf margins dry or die. Many viruses are also reported to cause necrosis of leaves, petioles and stems, they are tobacco mosaic virus, mosaic virus, cucumber mosaic virus, tobacco ring spot virus, yellow vein mosaic, etc.

Controlling techniques: Chemical fumigation of the soil, fungicide, bactericide, pruning, proper water and fertilizer management, good sanitation, heat treatment of planting stock, cut and remove the infected parts, genetically manipulating the plants for producing plants to resist fungi and bacteria are practices that are used to prevent or minimize the effects produced by microbes.

Insects

Ants, they are of different varieties, Argentine ant: Linepithema humile, Gray ants: Formica aerata and Formica perpilosa, Pavement ant: Tetramorium caespitum., Southern fire ant: Solenopsis xyloni, Thief ant: Solenopsis molesta, they spoil the soil by making nest and they feed honey dew secreted in plants.

Branch and Twig Borer (Melalgus confertus) burrow into the canes through the base of the bud or into the crotch formed by the shoot and spur. Feeding is often deep enough to completely conceal the adult in the hole. When shoots reach a length of 10–12 inches, a strong wind can cause the infected parts to twist and break. The click beetle (Limonius canus) can feed on buds. Cutworms (Peridroma saucia) (Amathes c-nigrum) (Orthndes rufula) injures the buds and so the buds may not develop. Leafhoppers (Erythroneura elegantuhi) (Erythroneum variabilis) remove the contents of leaf cells, leaving behind empty cells that appear as pale yellow spots.

Oak twig pruners (Anelaphiis spp. Linsley) are known as shoot, twig and root insects that affects the above mentioned parts.

Controlling techniques: Tilling the soil will also affects the nesting sites of ants and help to reduce their populations, collection and destruction of eggs, larvae, pupae and adults of insects, trapping the insects, insecticides, creating a situation to compete among males for mating with females, cutworms can be prevented by natural enemies like, predaceous or parasitic insects, mammals, parasitic nematodes, pathogens, birds, and reptiles,

Non insect pests

They are divided in to vertebrates and invertebrates. Vertebrates that disrupt the plants are monkeys, rats, birds, squirrels, etc. Non vertebrates are, Webspinning Spider Mites (Tetranychuspacificus) (Eotetranychus willamettei) (Tetranychus urticae) causes discoloration in leaves and yellow spots. Nematodes (Meloidogyne incognita) (Xiphinema americanutri) (Criconemella xenoplax) produces giant cell formation, disturbs the uptake of nutrients and water, and interferes with plant growth, crabs, snails are the other few invertebrates that causes trouble to the plant.

Controlling techniques: Construction of concrete ware houses, traps, biological methods, rodenticides, etc.

Weeds

Weeds reduce growth and yields of plants by competing for water, nutrients, and sunlight. Weed control enhances the establishment of new plants and improves the growth and yield of established plants. The skilled persons have many weed management tools available to achieve these objectives; however, the methods of using these tools vary from year to year and from place to place.

Soil characteristics are important to weed management. Soil texture and organic matter influence the weed species that are present, the number and timing of cultivations required, and the activity of herbicides. Annual species, such as puncturevine, crabgrass, horseweed, and Panicum spp., or perennials like johnsongrass, nutsedge, and bermudagrassare more prevalent on light-textured soil while perennials such as curly dock, field bindweed, and dallisgrass are more common on heavier-textured soils. Less preemergent herbicide is required for weed control on sandy, light soils, but residual control may be shorter than on clay or clay loam soils. Use low rates of herbicide on sandy soils or those low in organic matter. Clay soils are slower to dry for effective cultivation than sandy loam soils; thus, more frequent cultivation is practiced on lighter soils than heavy soils.

Few common weeds are, Bermudagrass, It is a vigorous spring- and summer-growing perennial. It grows from seed but its extensive system of rhizomes and stolons can also be spread during cultivation, Dallisgrass, It is a common perennial weed that can be highly competitive in newly planted plants; in established plants area it competes for soil moisture and nutrients. Dallisgrass seedlings germinate in spring and summer, and form new plants on short rhizomes that developed from the original root system. The other weeds are pigweeds Amaranthus spp. pineapple-weed Chamomilla suaveolens, nightshades Solanum spp., etc.

Apart from these, Parasitic and Epiphytic Plants like dodder (Cuscuta spp. L.), mistletoe (Phoradendron spp. Nutt.), American squawroot (Conopholis americana), etc., too affects the growth of plants,

Controlling techniques: Use of low rates of herbicides: Herbicides are traditionally discussed as two groups: those that are active against germinating weed seeds (preemergent herbicides) and those that are active on growing plants (postemergent herbicides). Some herbicides have both pre-and postemergent activity. Herbicides vary in their ability to control different weed species.

Preemergent herbicides are active in the soil against germinating weed seedlings. These herbicides are applied to bare soil and are leached into the soil with rain or irrigation where they affect germinating weed seeds. If herbicides remain on the soil surface without incorporation, some will degrade rapidly from sunlight. Weeds that emerge while the herbicide is on the surface, before it is activated by rain or irrigation, will not be controlled. Postemergent herbicides are applied to control weeds already growing in the vineyard. They can be combined with preemergent herbicides or applied as spot treatments during the growing season. In newly planted plants, selective postemergent herbicides are available for the control of most annual and perennial grasses, but not broadleaf weeds.

Frequent wetting of the soil promotes more rapid herbicide degradation in the soil. Herbicide degradation is generally faster in moist, warm soils than in dry, cold soils.

General Methods of Pest Controls

Other Factors that Affect the Cultivated Plants

Air Pollution

• Chemical discharges into the atmosphere have increased dramatically during this century, but the total effect on plants is virtually unknown. It has been demonstrated that air pollutants can cause mortality and losses in growth of plants. Nearly all species of deciduous and coniferous trees are sensitive to some pollutants. There are many chemicals released into the atmosphere singly and as compounds. In addition, other compounds are synthesized in the atmosphere. Some chemicals can be identified through leaf tissue analysis and by analysing the air. Generally, pollution injury first appears as leaf injury. Spots between the veins, leaf margin discoloration, and tip burns are common. These symptoms can also be influenced by host sensitivity, which is effected by genetic characteristics and environmental factors.

Herbicide

• Herbicides should be handled very carefully; misapplication of herbicides can often damage nontarget plants. The total extent of such damage remains unclear, but localized, severe damage occurs. Symptoms of herbicide injury are variable due to chemical mode of action, dosage, duration of exposure, plant species, and environmental conditions. Some herbicides cause growth abnormalities such as cupping or twisting of foliage while others cause foliage yellowing or browning, defoliation, or death.

PLANT HORMONES AND GROWTH REGULATORS

Plant hormones (phytohormones) are physiological intercellular messengers that control the complete plant lifecycle, including germination, rooting, growth, flowering, fruit ripening, foliage and death. In addition, plant hormones are secreted in response to environmental factors such asexcess of nutrients, drought conditions, light, temperature and chemical or physical stress. So, levels of hormones will change over the lifespan of a plant and are dependent upon season and environment.

The term ‘plant growth factor’ is usually employed for plant hormones or substances of similar effect that are administered to plants. Growth factors are widely used in industrialized agriculture to improve productivity. The application of growth factors allows synchronization of plant development to occur. For instance, ripening fruits can be controlled by setting desired atmospheric ethylene levels. Using this method, fruits that are separated from their parent plant will still respond to growth factors; allowing commercial plants to be ripened in storage during and after transportation. This way the process of harvesting can be run much more efficiently and effectively. Other applications include rooting of seedlings or the suppression of rooting with the simultaneous promotion of cell division as required by plant cell cultures. Just like with animal hormones, plant growth factors come in a wide variety, producing different and often antagonistic effects. In short, the right combination of hormones is vital to achieve the desired behavioural characteristics of cells and the productive development of plants as a whole. The plant growth regulators are classified into synthetic and native. The synthetic regulators are also known as exogenous regulators and the native are called the endogenous,

Five major classes of plant hormones are mentioned: auxins, cytokinins, gibbereilins, abscisic acid and ethylene. However as research progresses, more active molecules are being found and new families of regulators are emerging; one example being polyamines (putrescine or spermidine). Plant growth regulators have made the way for plant tissue culture techniques, which were a real boon for mankind in obtaining therapeutically valuable secondary metabolites.

Auxins

The term auxin is derived from the Greek word auxein which means to grow. Generally compounds are considered as auxins if they are able to induce cell elongation in stems and otherwise resemble indoleacetic acid (the first auxin isolated) in physiological activity. Auxins usually affect other processes in addition to cell elongation of stem cells but this characteristic is considered critical of all auxins and thus ‘helps’ define the hormone.

Auxins were the first plant hormones discovered. Charles Darwin was among the first scientists to pool in planthormone research. He described the effects of light on movement of canary grass coleoptiles in his book ‘The Power of Movement in Plants’ presented in 1880. The coleoptile is a specialized leaf originating from the first node which sheaths the epicotyl in the plants seedling stage protecting it until it emerges from the ground. When unidirectional light shines on the coleoptile, it bends in the direction of the light. If the tip of the coleoptile was covered with aluminium foil, bending would not occur towards the unidirectional light. However if the tip of the coleoptile was left uncovered but the portion just below the tip was covered, exposure to unidirectional light resulted in curvature toward the light. Darwin’s experiment suggested that the tip of the coleoptile was the tissue responsible for perceiving the light and producing some signal which was transported to the lower part of the coleoptile where the physiological response of bending occurred. When he cut off the tip of the coleoptile and exposed the rest of the coleoptile to unidirectional light curvature did not occur confirming the results of his experiment.

Salkowski (1885) discovered indole-3-acetic acid (IAA) in fermentation media. The isolation of the same product from plant tissues would not be found in plant tissues for almost 50 years. IAA is the major auxin involved in many of the physiological processes in plants. Fitting in 1907 put his efforts in studying signal transaction by making incisions on the light or dark side of the plant. He failed because the signal was capable of crossing or going around the incision, In 1913, modification was made in Fitting’s experiment by Boysen-Jensen, in that they inserted pieces of mica to block the transport of the signal and showed that transport of auxin toward the base occurs on the dark side of the plant as opposed to the side exposed to the unidirectional light. In 1918, Paal confirmed Boysen-Jensen’s results by cutting off coleoptile tips in the dark, exposing only the tips to the light, replacing the coleoptile tips on the plant but off centered to one side or the other. Results showed that whichever side was exposed to the coleoptile, curvature occurred toward the other side. Soding 1925, followed Paal’s idea and showed that if tips were cut off there was a reduction in growth but if they were cut off and then replaced growth continued to occur.

In 1926, Fritz Went reported a plant growth substance, isolated by placing agar blocks under coleoptile tips for a period of time then removing them and placing them on decapitated Avena stems. After placement over the agar, the stems resumed growth. In 1928, again Went developed a method of quantifying this plant growth substance. His results suggested that the curvatures of stems were proportional to the amount of growth substance in the agar. This test was called the avena curvature test. Much of our current knowledge of auxin was obtained from its applications. It was Went’s work, which had a great influence in stimulating plant growth substance research. He is often credited with dubbing the term auxin but it was actually Kogl and Haagen-Smit who purified the compound auxentriolic acid (auxin A) from human urine in 1931. Later Kogl isolated other compounds from urine which were similar in structure and function to auxin A. One of which was indole-3 acetic acid (IAA) initially discovered by Salkowski in 1885. In 1954 a committee of plant physiologists was set up to characterize the group auxins.

Indole acetic acid (IAA) is the principle natural auxin and other natural auxins are indole-3-acetonitrile (IAN), phenyl acetic acid and 4-chloroindole-3-acetic acid. The exogenous or synthetic auxins are indole-3-butyric acid (IBA), α-napthyl acetic acid (NAA), 2-napthyloxyacetic acid (NOA), 1-napthyl acetamide (NAD), 5-carboxymethyl-N, N-dimethyl dithiocarbonate, 2,4-dichlorophenoxy acetic acid (2,4-D), etc.

Production and occurrence

• Produced in shoot and root meristematic tissue, in young leaves, mature root cells and small amounts in mature leaves. Transported throughout the plant parts and the production of IAA will be more in day time. It is released by all cells when they are experiencing conditions which would normally cause a shoot meristematic cell to produce auxin. Ethylene has direct or indirect action over to enhance the synthesis auxin.

• IAA is chemically similar to the amino acid tryptophan which is generally accepted to be the molecule from which IAA is derived. Three mechanisms have been suggested to explain this conversion:

• Tryptophan is converted to indolepyruvic acid through a transamination reaction. Indolepyruvic acid is then converted to indoleacetaldehyde by a decarboxylation reaction. The final step involves oxidation of indoleacetaldehyde resulting in indoteacetic acid. 

• Tryptophan undergoes decarboxylation resulting in tryptamine. Tryptamine is then oxidized and deaminated to produce indoleacetaldehyde. This molecule is further oxidized to produce indoleacetic acid. 

• IAA can be produced via a tryptophan-independent mechanism. This mechanism is poorly understood, but has been proven using tip (-) mutants. Other experiments have shown that, in some plants, this mechanism is actually the preferred mechanism of IAA biosynthesis.

The enzymes responsible for the biosynthesis of IAA are most active in young tissues such as shoot apical meristems and growing leaves and fruits. These are the same tissues where the highest concentrations of IAA are found. One way plants can control the amount of IAA present in tissues at a particular time is by controlling the biosynthesis of the hormone. Another control mechanism involves the production of conjugates which are, in simple terms, molecules which resemble the hormone but are inactive. The formation of conjugates may be a mechanism of storing and transporting the active hormone. Conjugates can be formed from IAA via hydrolase enzymes. Conjugates can be rapidly activated by environmental stimuli signaling a quick hormonal response. Degradation of auxin is the final method of controlling auxin levels. This process also has two proposed mechanisms outlined below:

The oxidation of IAA by oxygen resulting in the loss of the carboxyl group and 3-methyleneoxindole as the major breakdown product. IAA oxidase is the enzyme which catalyses this activity. Conjugates of IAA and synthetic auxins such as 2,4-D can not be destroyed by this activity.

C-2 of the heterocyclic ring may be oxidized resulting in oxindole-3-acetic acid. C-3 may be oxidized in addition to C-2 resulting in dioxindole-3-acetic acid. The mechanisms by which biosynthesis and degradation of auxin molecules occur are important to future agricultural applications. Information regarding auxin metabolism will most likely lead to genetic and chemical manipulation of endogenous hormone levels resulting in desirable growth and differentiation of important plant species.

Functions of auxin

Stimulates cell elongation. 

The auxin supply from the apical bud suppresses growth of lateral buds. Apical dominance is the inhibiting influence of the shoot apex on the growth of axillary buds. Removal of the apical bud results in growth of the axillary buds. Replacing the apical bud with a lanolin paste containing IAA restores the apical dominance. The mechanism involves another hormone - ethylene. Auxin (IAA) causes lateral buds to make ethylene, which inhibits growth of the lateral buds.

• Differentiation of vascular tissue (xylem and phloem) is stimulated by IAA.

• Auxin stimulates root initiation on stem cuttings and lateral root development in tissue culture (adventitious rooting).

• Auxin mediates the tropistic response of bending in response to gravity and light (this is how auxin was first discovered).

• Auxin has various effects on leaf and fruit abscission, fruit set, development, and ripening, and flowering, depending on the circumstances.

Cytokinins

Cytokinins are compounds with a structure resembling adenine which promote cell division and have other similar functions to kinetin. They also regulate the pattern and frequency of organ production as well as position and shape. They have an inhibitory effect on senescence. Kinetin was the first cytokinin identified and so named because of the compounds ability to promote cytokinesis (cell division). Though it is a natural compound, it is not made in plants, and is therefore usually considered a ‘synthetic’ cytokinin. The common naturally occurring cytokinin in plants today is called zeatin which was isolated from corn.

Cytokinin have been found in almost all higher plants as well as mosses, fungi, bacteria, and also in many prokaryotes and eukaryotes. There are more than 200 natural and synthetic cytokinins identified. Cytokinin concentrations are more in meristematic regions and areas of continuous growth potential such as roots, young leaves, developing fruits, and seeds.

Haberlandt (1913) and Jablonski and Skoog (1954) identified that a compound found in vascular tissues had the ability to stimulate cell division. In 1941, Johannes van Overbeek discovered that the milky endosperm from coconut and other various species of plants also had this ability. The first cytokinin was isolated from herring sperm in 1955 by Miller and his associates. This compound was named kinetin because of its ability to promote cytokinesis (cell division). The first naturally occurring cytokinin was isolated from corn in 1961 by Miller and it was later called zeatin. Since that time, many more naturally occurring cytokinins have been isolated and the compound was common to all plant species in one form or another.

The naturally occurring cytokinin's are zeatin, N6 dimethyl amino purine, isopentenyl aminopurine. The synthetic cytokinin's are kinetin, adenine, 6-benzyl adenine benzimidazole and N, N’-diphenyl urea.

Production and occurrence

Produced in root and shoot meristematic tissue, in mature shoot cells and in mature roots in small amounts. If is rapidly transported in xylem stream. Peak production occurs in daytime and their activity is reduced in plants suffering drought. It is directly or indirectly induced by high levels of Gibberellic acid.

Cytokinin is generally found in meristematic regions and growing tissues. They are believed to be synthesized in theroots and translocated via the xylem to shoots. Cytokinin biosynthesis happens through the biochemical modification of adenine. They are synthesized by following pathway

A product of the mevalonate pathway called isopentyl pyrophosphate is isomerized. This isomer can then react with adenosine monophosphate with the aid of an enzyme called isopentenyl AMP synthase. The result is isopentenyl adenosine-5’-phosphate (isopentenyl AMP). This product can then be converted to isopentenyl adenosine by removal of the phosphate by a phosphatase and further converted to isopentenyl adenine by removal of the ribose group. Isopentenyl adenine can be converted to the three major forms of naturally occurring cytokinin's.

Other pathways or slight alterations of this one probably leads to the other forms. Degradation of cytokinin's occurs largely due to the enzyme cytokinin oxidase. This enzyme removes the side chain and releases adenine. Derivatives can also be made but the difficulties are with pathways, which are more complex and poorly understood.

Functions of cytokinin

• Stimulate cell division (cytokinesis).

• Stimulate morphogenesis (shoot initiation/bud formation) in tissue culture.

• Stimulate the growth of lateral (or adventitious) buds release of apical dominance.

• Stimulate leaf expansion resulting from cell enlargement.

• May enhance stomatal opening in some species (Figure 6.2).

• Promotes the conversion of etioplasts into chloroplasts via

• stimulation of chlorophyll synthesis. Stimulate the dark-germination of light-dependent seeds.

• Delays senescence.

• Promotes some stages of root development. 

Effect of cytokinin on stomatal opening 

Ethylene

Ethylene has been used in practice since the ancient times, where people would use gas figs in order to stimulate ripening, burn incense in closed rooms to enhance the ripening of pears. It was in 1864, that leaks of gas from streetlights showed stunting of growth, twisting of plants, and abnormal thickening of stems. In 1901, a Russian scientist named Dimitry Neljubow showed that the active component was ethylene. Doubt 1917 discovered that ethylene stimulated abscission. In 1932 it was demonstrated that the ethylene evolved from stored apple inhibited the growth of potato shoots enclosed with them. In 1934 Gane reported that plants synthesize ethylene. In 1935, Crocker proposed that ethylene was the plant hormone responsible for fruit ripening as well as inhibition of vegetative tissues. Ethylene is now known to have many other functions as well.

Production and occurrence 

• Production is directly induced by high levels of Auxin, root flooding and drought. It is found in germinating seeds and produced in nodes of stems, tissues of ripening fruits, response to shoot environmental, pest, or disease stress and in senescent leaves and flowers. Light minimizes the production of ethylene. It is released by all cells when they are experiencing conditions which would normally cause a mature shoot cell to produce ethylene.

• Ethylene is produced in all higher plants and is produced from methionine in essentially all tissues. Production of ethylene varies with the type of tissue, the plant species, and also the stage of development. The mechanism by which ethylene is produced from methionine is a three-step process. ATP is an essential component in the synthesis of ethylene from methionine. ATP and water are added to methionine resulting in loss of the three phosphates and S-adenosyl methionine (SAM). 1-amino-cyclopropanel-carboxylic acid synthase (ACC-synthase) facilitates the production of ACC from SAM. Oxygen is then needed in order to oxidize ACC and produce ethylene. This reaction is catalyzed by an oxidative enzyme called ethylene forming enzyme. The control of ethylene production has received considerable study. Study of ethylene has focused around the synthesis promoting effects of auxin, wounding, and drought as well as aspects of fruit-ripening. ACC synthase is the rate limiting step for ethylene production and it is this enzyme that is manipulated in biotechnology to delay fruit ripening in the ‘flavor saver’ tomatoes.

Functions of ethylene

• Production stimulated during ripening, flooding, stress, senescence, mechanical damage, infection. 

• Regulator of cell death programs in plants (apoptosis). 

• Stimulates the release of dormancy. 

• Stimulates shoot and root growth and differentiation (triple response). 

• Regulates ripening of climacteric fruits. 

• May have a role in adventitious root formation. 

• Stimulates leaf and fruit abscission. 

• Flowering in most plants is inhibited by ethylene. Mangos, pineapples and some ornamentals are stimulated by ethylene.

• Induction of femaleness in dioecious flowers. 

• Stimulates flower opening. 

• Stimulates flower and leaf senescence.

Gibberellins

Unlike the classification of auxins which are classified on the basis of function, gibberellins are classified on the basis of structure as well as function. All gibberellins are derived from the ent-gibberellane skeleton. The gibberellins are named GA1 . GAn in order of discovery. Gibberellic acid was the first gibberellin to be structurally characterized as GA3 . There are currently 136 GAs identified from plants, fungi and bacteria.

• They are a group of diterpenoid acids that functions as plant growth regulators influencing a range of developmental processes in higher plants including stem elongation, germination, dormancy, flowering, sex expression, enzyme induction and leaf and fruit senescence. The origin of research into gibberellins can be traced to Japanese plant pathologists who were investigating the causes of the ‘bakanae’ (foolish seedling) disease which seriously lowered the yield of rice crops in Japan, Taiwan and throughout the Asian countries. Symptoms of the disease are pale yellow, elongated seedlings with slender leaves and stunted roots. Severely diseased plants die whereas plants with slight symptoms survive but produce poorly developed grain, or none at all.

• Bakanae is now easily prevented by treatment of seeds with fungicides prior to sowing. In 1898 Shotaro Hori demonstrated that the symptoms were induced by infection with a fungus belonging to the genus Fusarium, probably Fusarium heterosporium Necs.

• In 1912, Sawada suggested that the elongation in riceseedlings infected with bakanae fungus might be due to a stimulus derived from fungal hyphae.

• Subsequently, Eiichi Kurosawa (1926) found that culture filtrates from dried rice seedlings caused marked elongation in rice and other sub-tropical grasses. He concluded that bakanae fungus secretes a chemical that stimulates shoot elongation, inhibits chlorophyll formation and suppresses root growth.

• Although there has been controversy among plant pathologists over the nomenclature of bakanae fungus, in the 1930s, the imperfect stage of the fungus was named Fusarium moniliforme (Sheldon) and the perfect stage, was named as Gibberella fujikuroi (Saw.) Wr. by H.W. Wollenweber. The terms ‘Fujikuroi’ and ‘Saw’ in Gibberella fujikuroi (Saw.) Wr. were derived from the names of two distinguished Japanese plant pathologists, Yosaburo Fujikuro and Kenkichi Sawada.

• In 1934, Yabuta isolated a crystalline compound from the fungal culture filtrate that inhibited growth of rice seedlings at all concentrations tested. The structure of the inhibitor was found to be 5-n-butylpicolinic acid or fusaric acid. The formation of fusaric acid in culture filtrates was suppressed by changing the composition of the culture medium. As a result, a noncrystalline solid was obtained from the culture filtrate that stimulated the growth of rice seedlings. This compound was named gibberellin by Yabuta.

• In 1938, Yabuta and his associate Yusuke Sumiki finally succeeded in crystallizing a pale yellow solid to yield gibberellin A and gibberellin B (The names were subsequently interchanged in 1941 and the original gibberellin A was found to be inactive.) Determination of the structure of the active gibberellin was hampered by a shortage of pure crystalline sample. In the United States, the first research on gibberellins began after the Second World War. In 1950, John E. Mitchell reported optimal fermentation procedures for the fungus, as well as the effects of fungal extracts on the growth of bean (Vicia faba) seedlings. In Northern USDA Regional Research Laboratories in Peoria, large scale fermentations were carried out with the purpose of producing pure gibberellin A for agricultural uses but initial fermentations were found to be inactive. Further researches were carried out by Sumiki in 1951, Stodola et al., 1955, Curtis and Cross, 1954 regarding gibberellins and finally the gibberllic acid was determined by its chemical and physical properties.

• In 1955, members of Sumuki group, succeeded in separating the methyl ester of gibberellin A into three components, from which corresponding free acids were obtained and named gibberellins Al, A2, and A3. Gibberellin A3 was found to be identical to gibberellic acid. In 1957, Takahashi et al. isolated a new gibberellin named gibberellin A4 as a minor component from the culture filtrate.

• In the mid 1950s, evidence that gibberellins were naturally occurring substances in higher plants began to appear in the literature. Margaret Radley in the UK demonstrated the presence of gibberellin-like substances in higher plants. In the United States, Bernard Phinney et al were the first to report gibberellin-like substance in maize. This was followed by the isolation of crystalline gibberellin Al, A5, A6 and A8 from runner bean (Phaseotus multiflorus). After 10 years the number of gibberellins reported in the literature isolated from fungal and plant origins rapidly increased. In 1968, J. MacMillan and N. Takahashi concluded that all gibberellins should be assigned numbers as gibberellin A1-x, irrespective of their origin. Over the past 20 years using modern analytical techniques many more gibberellins have been identified. At the present time the number of gibberellins identified is 126.

Production and occurrence

• Produced in the roots, embryo and germinating seeds. The level of gibberellins goes up in the dark when sugar cannot be manufactured and will be reduced in the light. It is released in mature cells (particularly root) when they do not have enough sugar and oxygen to support both themselves and released by all cells when they are experiencing conditions which would normally cause a mature root cell to produce GA.

• Gibberellins are diterpenes synthesized from acetyl CoA via the mevalonic acid pathway. They all have either 19 or 20 carbon units grouped into either four or five ring systems. The fifth ring is a lactone ring as shown in the structures above attached to ring A. Gibberellins are believed to be synthesized in young tissues of the shoot and also the developing seed. It is not clear whether young root tissues also produce gibberellins. There is also some evidence that leaves may also contain them. The gibberellins are formed through the pathway, three acetyl CoA molecules are oxidized by two NADPH molecules to produce three CoA molecules as a side product and mevalonic acid. Mevalonic acid is then Phosphorylated by ATP and decarboxylated to form isopentyl pyrophosphate. Four of these molecules form geranylgeranyl pyrophosphate which serves as the donor for all GA carbon atoms.

• This compound is then converted to copalylpyrophosphate which has 2 ring systems. Copalylpyrophosphate is then converted to kaurene which has 4-ring systems. Subsequent oxidations reveal kaurenol (alcohol form), kaurenal (aldehyde form), and kaurenoic acid respectively

• Kaurenoic acid is converted to the aldehyde form of GA12 by decarboxylation. GA12 is the first true gibberellane ring system with 20 carbons. From the aldehyde form of GA12 arise both 20 and 19 carbon gibberellins but there are many mechanisms by which these other compounds arise. During active growth, the plant will metabolize most gibberellins by hydroxylation to inactive conjugates quickly with, the exception of GA3. GA3 is degraded much slower which helps to explain why the symptoms initially associated with the hormone in the disease bakanae are present. Inactive conjugates might be stored or translocated via the phloem and xylem before their release (activation) at the proper time and in the proper tissue.

Functions of gibberellins

• Stimulates stem elongation by stimulating cell division and elongation. GA controls internode elongation in the mature regions of plants. Dwarf plants do not make enough active forms of GA. 

• Flowering in biennial plants is controlled by GA. Biennials grow one year as a rosette and after the winter, they bolt (rapid expansion of internodes and formation of flowers).

• Breaks seed dormancy in some plants that require stratification or light to induce germination. 

• Stimulates α-amylase production in germinating cereal grains for mobilization of seed reserves. 

• Juvenility refers to the different stages that plants may exist in. GA may help determine whether a particular plant part is juvenile or adult. 

• Stimulates germination of pollen and growth of pollen tubes. 

• Induces maleness in dioecious flowers (sex expresion). 

• Can cause parthenocarpic (seedless) fruit development or increase the size of seedless fruit (grapes). 

• Can delay senescence in leaves and citrus fruits. 

• May be involved in phytochrome responses.

Abscisic Acid

Natural growth inhibiting substances are present in plants and affect the normal physiological process of them. One such compound is abscisic acid, a single compound unlike the auxins, gibberellins, and cytokinins. It was called ‘abscisin II’ originally because it was thought to play a major role in abscission of fruits. At about the same time another group was calling it ‘dormin’ because they thought it had a major role in bud dormancy. Though abscisic acid generally is thought to play mostly inhibitory roles, it has many promoting functions as well.

In 1963, when Frederick Addicott and his associates were the one to identify abscisic acid. Two compounds were isolated and named as abscisin I and abscisin II. Abscisin II is presently called abscisic acid (ABA). At the same time Philip Wareing, who was studying bud dormancy in woody plants and Van Steveninck, who was studying abscission of flowers and fruits discovered the same compound.

Production and occurrence

• ABA is a naturally occurring sesquiterpenoid (15-carbon) compound in plants, which is partially produced via the mevalonic pathway in chloroplasts and other plastids. Because it is synthesized partially in the chloroplasts, it makes sense that biosynthesis primarily occurs in the leaves. The production of ABA is by stresses such as water loss and freezing temperatures. The biosynthesis occurs indirectly through the production of carotenoids. Breakdown of these carotenoids occurs by the following mechanism: Violaxanthin (forty carbons) is isomerized and then splitted via an isomerase reaction followed by an oxidation reaction. One molecule of xanthonin is produced from one molecule of violaxanthonin and it is not clear what happens to the remaining byproducts. The one molecule of xanthonin produced is unstable and spontaneously changed to ABA aldehyde. Further oxidation results in ABA. Activation of the molecule can occur by two methods. In the first, method, an ABA-glucose ester can form by attachment of glucose to ABA. In the second method, oxidation of ABA can occur to form phaseic acid and dihyhdrophaseic acid. Both xylem and phloem tissues carries ABA. It can also be translocated through parenchyma cells. Unlike auxins, ABA is capable of moving both up and down the stem.

Functions of abscisic acid 

• The abscisic acid stimulates the closure of stomata (water stress brings about an increase in ABA synthesis) 

• Involved in abscission of buds, leaves, petals, flowers, and fruits in many, if not all, instances, as well as in dehiscence of fruits. 

• Production is accentuated by stresses such as water loss and freezing temperatures. 

• Involved in bud dormancy. 

• Prolongs seed dormancy and delays germination (vivipary). 

• Inhibits elongation. 

• ABA is implicated in the control of elongation, lateral root development, and geotropism, as well as in water uptake and ion transport by roots. 

• ABA coming from the plastids promotes the metabolism of ripening. 

• Promotes senescence. 

• Can reverse the effects of growth stimulating hormones

Closure of stomata and water stress brings about an increase in ABA synthesis

Polyamines

• Polyamines are unique as they are effective in relatively high concentrations. Typical concentrations range from 5 to 500 mg/L. Polyamines influence flowering and promote plant regeneration. Few examples are Spermine, Spermidine and Putrescine. They play a major role in basic genetic processes such as DNA synthesis and gene expression. Spermine and spermidine bind to the phosphate backbone of nucleic acids. The interaction is mostly based on electrostatic interactions between negatively charged phosphates of the nucleic acids and the positively charged ammonium groups of the polyamines.

• Polyamines are responsible for cell migration, proliferation and differentiation in plants. They represent a group of plant growth hormones, but they also have an effect on skin, hair growth, female fertility, fat depots, pancreatic integrity and regenerative growth in mammals. In addition, spermine is an important reagent widely used to precipitate DNA in molecular biology protocols. Spermidine is a standard reagent in PCR applications.

• Spermine and spermidine are derivatives of putrescine (1,4-diaminobutane) which is produced from L-ornithine by action of ODC (ornithine decarboxylase). L-ornithine is the product of L-arginine degradation by arginase. Spermidine is a triamine structure that is produced by spermidine synthase (SpdS) which catalyses monoalkylation of putrescine (1,4-diaminobutane) with decarboxylated S-adenosylmethionine (dcAdoMet) 3-aminopropyl donor. The formal alkylation of both amino groups of putrescine with the 3-aminopropyl donor yields the symmetrical tetraamine spermine.

Brassinosteroids

• There are approximately 60 naturally occurring polyhydroxy steroids known as brassinosteroids (BRs). They are named after the first one identified, brassinolide, which was isolated from rape in 1979. They appear to be widely distributed in the plant kingdom.

• In the early 1980s USDA scientists showed that BR could increase yields of radishes, lettuce, beans, peppers and potatoes. However, subsequent results under field conditions were disappointing because inconsistent results were obtained. For this reason testing was phased out in the United States. More recently large-scale field trials in China and Japan over a six-year period have shown that 24-epibrassinolide, an alternative to brassinolide, increased the production of agronomic and horticultural crops (including wheat, corn, tobacco, watermelon, and cucumber). However, once again depending on cultural conditions, method of application, and other factors, the results sometimes were striking while other times they were marginal. Further improvements in the formulation, application method, timing, effects of environmental conditions, and other factors need tobe investigated further in order to identify the reason for these variable results.

• Brassinosteroids may be a new class of plant growth substances. They are widely distributed within the plant kingdom, they have an effect at extremely low concentrations, both in bioassays and whole plants, and they have a range of effects that are different from the other classes of plant substances. Finally, they can be applied to one part of the plant and transported to another location where, in very low amounts, they elicit a biological response.

Functions of brassinosteroids

•  Promote shoot elongation at low concentrations. 

•  Strongly inhibit root growth and development. 

•  Promote ethylene biosynthesis and epinasty. 

•  Interfere with ecdysteroids (moulting hormones) in insects. 

• Have had contradictory effects in tissue culture. 24-epibrassinolide has been shown to mimic culture conditioning factors and to be synergistic with these factors in promoting carrot cell growth. However, in transformed tobacco cells brassinosteroids in low concentrations significantly inhibited cell growth. 

• Enhance xylem differentiation. 

• Decrease fruit abortion and drop. 

• Enhance resistance to chilling, disease, herbicide, and salt stress. 

• Promotion of germination. 

• Promote changes in plasmalemma energization and transport, assimilate uptake. 

• Increase RNA and DNA polymerase activities and synthesis of RNA, DNA, and protein.

Salicylic Acid

• Salicylic acid has been known to be present in some plant tissues for quite some time, but has only recently been recognized as a potential PGR. Salicylic acid is synthesized from the amino acid phenylalanine. SA is thought by some to be a new class of plant growth regulator. It is a chemically characterized compound, ubiquitously found in the plant kingdom and has an effect on many physiological processes in plants at low concentrations. Further molecular studies on SA signal transduction should yield insights into the mechanism of action of this important regulatory compound.

Functions of salicylic acid

• Promotes flowering. 

• Stimulates thermogenesis in Arum flowers. 

• Stimulates plant pathogenesis protein production (systemic acquired resistance). 

• May enhance longevity of flowers. 

• May inhibit ethylene biosynthesis. 

• May inhibit seed germination.

• Blocks the wound response. 

• Reverses the effects of ABA.

Jasmonates

• Jasmonates are represented by jasmonic acid and its methyl ester. They were first isolated from the jasmine plant in which the methyl ester is an important product in the perfume industry. Jasmonic acid is synthesized from linolenic acid, which is an important fatty acid. Jasmonic acid is considered by some to be a new class of plant growth regulator. It is a chemically characterized compound and has been identified in many plant species. It has physiological effects at very low concentrations and indirect evidence suggests that it is transported throughout the plant.

Functions of jasmonates

• Inhibition of many processes such as seedling longitudinal growth, root length growth, mycorrhizial fungi growth, tissue culture growth, embryogenesis, seed germination, pollen germination, flower bud formation, carotenoid biosynthesis, chlorophyll formation, rubisco biosynthesis, and photosynthetic activities 

• Promotion of senescence, abscission, tuber formation, fruit ripening, pigment formation, tendril coiling, differentiation in plant tissue culture, adventitious root formation, breaking of seed dormancy, pollen germination, stomatal closure, microtubule disruption, chlorophyll degradation, respiration, ethylene biosynthesis, and protein synthesis 

• They play an important role in plant defense by inducing proteinase synthesis.

COLLECTION OF CRUDE DRUGS

• Collection is the most important step which comes after cultivation. Drugs are collected from wild or cultivated plants and the tasks for collection depends upon the collector, whether he is a skilled or unskilled labour. Drugs should be collected when they contain maximum amount of constituents in a highly scientific manner. The season at which each drug is collected is so important, as the amount, and sometimes the nature, of the active constituents could be changed throughout the year. For example, Rhubarb is collected only in summer seasons because no anthraquinone derivatives would be present in winter season but anthranols are converted to anthraquinones during summer. Not only the season but also the age of the plant should be taken in to great consideration since it governs not only the total amount of active constituents produced in the plants but also the proportions of the constituents of the active mixture. High proportion of pulegone in young plants of peppermint will be replaced by menthone and menthol and reduction in the percentage of alkaloids in datura as the plant ages are examples of the effect of aging in plants.

• Moreover, the composition of a number of secondary plant metabolites varies throughout the day and night, and it is believed that some inter conversion would happen during day and night.

• Generally, the leaves are collected just before the flowering season, e.g. vasaka, digitalis, etc., at this time it is assumed that the whole plant has come to a healthy state and contain an optimum amount of metabolites, flowers are collected before they expand fully, e.g. clove, saffron, etc., and underground organs as the aerial parts of plant cells die, e.g. liquorice, rauwolfia, etc. Since it is very difficult to collect the exact medicinally valuable parts, the official pharmacopoeia’s has fixed certain amount of foreign matter that is permissible with drug. Some fruits are collected after their full maturity while the others are collected after the fruits are ripe. Barks are usually collected in spring season, as they are easy to separate from the wood during this season. The barks are collected using three techniques, felling (bark is peeled off after cutting the tree at base), uprooting (the underground roots are dug out and barks are collected from branches and roots) and coppicing (plant is cut one metre above the ground level and barks are removed).

• Underground parts should be collected and shaken, dusted in order to remove the adhered soil; water washing could be done if the adhered particles are too sticky with plant parts. The unorganized drugs should be collected from plants as soon as they oozes out, e.g., resins, latex, gums, etc. Discoloured drugs or drugs which were affected by insects should be rejected.

HARVESTING OF CRUDE DRUGS

• Harvesting is an important operation in cultivation technology, as it reflects upon economic aspects of the crude drugs. An important point which needs attention over here is the type of drug to be harvested and the pharmacopoeial standards which it needs to achieve. Harvesting can be done efficiently in every respect by the skilled workers. Selectivity is of advantage in that the drugs other than genuine, but similar in appearance can be rejected at the site of collection. It is, however, a laborious job and may not be economical. In certain cases, it cannot be replaced by any mechanical means, e.g. digitalis, tea, vinca and senna leaves. The underground drugs like roots, rhizomes, tubers, etc. are harvested by mechanical devices, such as diggers or lifters. The tubers or roots are thoroughly washed in water to get rid of earthy-matter. Drugs which constitute all aerial parts are harvested by binders for economic reasons. Many a times, flowers, seeds and small fruits are harvested by a special device known as seed stripper. The technique of beating plant with bamboos is used in case of cloves. The cochineal insects are collected from branches of cactiby brushing. The seaweeds producing agar are harvested by long handled forks. Peppermint and spearmint are harvested by normal method with mowers, whereas fennel, coriander and caraway plants are uprooted and dried. After drying, either they are thrashed or beaten and the fruits are separated by winnowing. Sometimes, reaping machines are also used for their harvesting.

DRYING OF CRUDE DRUGS

• Before marketing a crude drug, it is necessary to process it properly, so as to preserve it for a longer time and also to acquire better pharmaceutical elegance. This processing includes several operations or treatments, depending upon the source of the crude drug (animal or plant) and its chemical nature. Drying consists of removal of sufficient moisture content of crude drug, so as to improve its quality and make it resistant to the growth of microorganisms. Drying inhibits partially enzymatic reactions. Drying also facilitates pulverizing or grinding of a crude drug. In certain drugs, some special methods are required to be followed to attain specific standards, e.g. fermentation in case of Cinnamomum zeylanicum bark and gentian roots. The slicing and cutting into smaller pieces is done to enhance drying, as in case of glycyrrhiza, squill and calumba. The flowers are dried in shade so as to retain their colour and volatile oil content. Depending upon the type of chemical constituents, a method of drying can be used for a crude drug. Drying can be of two types - (1) natural (sun drying) and (2) artificial.

Natural Drying (Sun-Drying)

• In case of natural drying, it may be either direct sun-drying or in the shed. If the natural colour of the drug (digitalis, clove, senna) and the volatile principles of the drug (peppermint) are to be retained, drying in shed is preferred. If the contents of the drugs are quite stable to the temperature and sunlight, the drugs can be dried directly in sunshine (gum acacia, seeds and fruits).

Artificial Drying

Drying by artificial means includes drying the drugs in 

• an oven; i.e. tray-dryers; 

• vacuum dryers and 

• spray dryers.

Tray dryers

• The drugs which do not contain volatile oils and are quite stable to heat or which need deactivation of enzymes are dried in tray dryers. In this process, hot air of the desired temperature is circulated through the dryers and this facilitates the removal of water content of the drugs (belladonna roots, cinchona bark, tea and raspberry leaves and gums are dried by this method).

Vacuum dryers

• The drugs which are sensitive to higher temperature are dried by this process, e.g. Tannic acid and digitalis leaves.

Spray dryers

• Few drugs which are highly sensitive to atmospheric conditions and also to temperature of vacuum-drying are dried by spray-drying method. The technique is followed for quick drying of economically important plant or animal constituents, rather than the crude drugs. Examples of spray drying are papaya latex, pectin, tannins, etc.

GARBLING (DRESSING)

• The next step in preparation of crude drug for market after drying is garbling. This process is desired when sand, dirt and foreign organic parts of the same plant, not constituting drug are required to be removed. This foreign organic matter (extraneous matter) is removed by several ways and means available and practicable at the site of the preparation of the drugs. If the extraneous matter is permitted in crude drugs, the quality of drug surfers and at times, it dose not pass pharmacopoeial limits. Excessive stems in case of lobelia and stramonium need to be removed, while the stalks, in case of cloves are to be deleted. Drugs constituting rhizomes need to be separated carefully from roots and rootlets and also stem bases. Pieces of iron must be removed with the magnet in case of castor seeds before crushing and by shifting in case of vinca and senna leaves. Pieces of bark should be removed by peeling as in gum acacia.

PACKING OF CRUDE DRUGS

• The morphological and chemical nature of drug, its ultimate use and effects of climatic conditions during transportation and storage should be taken into consideration while packing the drugs. Aloe is packed in goat skin. Colophony and balsam of tolu are packed in kerosene tins, while asafoetida is stored in well closed containers to prevent loss of volatile oil. Cod liver oil, being sensitive to sunlight, should be stored in such containers, which will not have effect of sunlight, whereas, the leaf drugs like senna, vinca and others are pressed and baled. The drugs which are very sensitive to moisture and also costly at the same time need special attention, e.g. digitalis, ergot and squill. Squill becomes flexible; ergot becomes susceptible to the microbial growth, while digitalis looses its potency due to decomposition of glycosides, if brought in contact with excess of moisture during storage. Hence, the chemicals which absorb excessive moisture (desiccating agents) from the drug are incorporated in the containers. Colophony needs to be packed in big masses to control autooxidation. Cinnamon bark, which is available in the form of quills, is packed one inside the other quill, so as to facilitate transport and to prevent volatilization of oil from the drug.

• The crude drugs like roots, seeds and others do not need special attention and are packed in gunny bags, while in some cases bags are coated with polythene internally. The weight of certain drugs in lots is also kept constant e.g. Indian opium.

STORAGE OF CRUDE DRUGS

• Preservation of crude drugs needs sound knowledge of their physical and chemical properties. A good quality of the drugs can be maintained, if they are preserved properly. All the drugs should be preserved in well closed and, possibly in the filled containers. They should be stored in the premises which are water-proof, fire proof and rodentproof. A number of drugs absorb moisture during their storage and become susceptible to the microbial growth. Some drugs absorb moisture to the extent of 25% of their weight. The moisture, not only increases the bulk of the drug, but also causes impairment in the quality of crude drug. The excessive moisture facilitates enzymatic reactions resulting in decomposition of active constituents e.g. digitalis leaves and wild cherry bark. Gentian and ergot receive mould infestation due to excessive moisture. Radiation due to direct sun-light also causes destruction of active chemical constituents, e.g. ergot, cod liver oil and digitalis. Form or shape of the drug also plays very important role in preserving the crude drugs. Colophony in the entire form (big masses) is preserved nicely, but if stored in powdered form, it gets oxidized or looses solubility in petroleum ether. Squill, when stored in powdered form becomes hygroscopic and forms rubbery mass on prolonged exposure to air. The fixed oil in the powdered ergot becomes rancid on storage. In order to maintain a good quality of ergot, it is required that the drug should be defatted with lipid solvent prior to storage. Lard, the purified internal fat of the abdomen of the hog, is to be preserved against rancidity by adding siam benzoin. Atmospheric oxygen is also destructive to several drugs and hence, they are filled completely in well closed containers, or the air in the container is replaced by an inert gas like nitrogen; e.g. shark liver oil, papain, etc.

• Apart from protection against adverse physical and chemical changes, the preservation against insect or mould attacks is also important. Different types of insects, nematodes, worms, moulds and mites infest the crude drugs during storage. Some of the more important pests found in drugs are Coleoptera (Stegobium paniceum and Calandrum granarium), Lepidoptera (Ephestia kuehniella and Tinea pellionella), and Archnida or mites (Pyroglyphids farinae and Glyophagus domesticus). They can be prevented by drying the drug thoroughly before storage and also by giving treatment of fumigants. The common fumigants used for storage of crude drugs are methyl bromide, carbon disulphide and hydrocyanic acid. At times, drugs are given special treatment, such as liming of the ginger and coating of nutmeg. Temperature is also very important factor in preservation of the drugs, as it accelerates several chemical reactions leading to decomposition of the constituents. Hence, most of the drugs need to be preserved at a very low temperature. The costly phytopharmaceuticals are required to be preserved at refrigerated temperature in well closed containers. Small quantities of crude drugs could be readily stored in airtight, moisture proof and light proof containers such as tin, cans, covered metal tins, or amber glass containers. Wooden boxes and paper bags should not be used for storage of crude drugs.

QUALITY MANAGEMENT

• The herbal drug manufacturers should establish a quality management department which is responsible for supervision and quality control for the entire production process, and should have adequate staff, premises, instruments and equipment to meet the standard requirements of the scale of production and species identification. The quality management department should monitor the environment and hygienic management, test production materials, packaging materials and the crude drugs, and issue testing reports, develop training plans and supervise their implementation; and also they should manage the original records of production, packaging, testing, etc. Prior to packaging, the quality control department should test each batch of the crude drugs in accordance with the national or approved standards for crude drugs. The testing procedures should include macroscopic characters and identification, impurities, moisture, ash and acid insoluble ash, extracts, and assay for marker or active constituents. Pesticide residue, heavy metals and microbiological limits should comply with the national standards and the relevant requirements. The testing reports should be signed by the operator and the responsible person of the quality control department, and then filed. As far as the personnel and facilities are concerned, they should possess qualifications of college education or above in pharmacy, knowledge in alternative systems of medicines, agronomy, animal husbandry orthel relevant specialties, trained on production techniques, safety, and hygiene and have experience in the production of crude drugs, quality management of crude drugs. Staff engaged in the field work should be familiar with cultivation techniques, especially the use of pesticides and safety protection; those engaged in rearing should be familiar with rearing techniques.

• The personnel engaged in processing, packaging or testing should undergo health examinations regularly and those suffering from infectious diseases, dermatitis or open wounds shall not be allowed to do work which is in direct contact with crude drugs. The producer should designate a person to be responsible for checking sanitation and hygiene. The applicable range and precision of instruments, meters, measures, weighers and balances, etc. used in production and testing, should conform to the relevant requirements, their performance status should be clearly indicated, and calibration should be conducted regularly. 

DOCUMENTATION

• The producer should maintain its standard operating procedures for production and quality management. Detailed records for the entire production process of each crude drug should be documented, and if necessary, photos or images might be attached, which should include, origin of seeds, strains and propagation materials, production techniques and process, sowing time, quantity and area of medicinal plants, seedling, transplantation, and the type, application schedule, quantity arid usage of fertilizer, quantity, application schedule and usage of pesticide, microbicide or herbicide, Collection time and yield, fresh weight and processing, drying and drying loss, transport and storage of medicinal parts. Quality evaluations of crude drugs: description of macroscopic characters of crude drugs and records of test results. All these records, production plans and their details, contracts or agreements etc. should be filed and kept properly by a designated person.