Effect of different Sugars on in vitro propagation of Dioscorea
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1. Tuber Crops
Tuber crops are the third most important food crops of man after cereals and grain legumes. These are a group of plants which produce underground plant parts which are storage in function. They are mainly classified into two:
1. Major tropical tuber crops
2. Minor tuber crops
The major tropical tuber crops grown in our country are cassava, sweet potato, yams & aroids. The minor tuber crops include coleus, Pachyrrhizus, arrow root etc. These tubers are rich source of carbohydrate and many of them have medicinal value.
Minor Tuber Crops
It is minor tuber crop. Out of the 200 spp of coleus, 8 species are recorded in India. Many species are ornamental, some are of medicinal value. Coleus parviflorus, commonly known as Chinese potato is the only edible species. It is grown in India, Srilanka, Java, Indo-China and parts of Tropical Africa.
Yam bean (Pachyrrhizus exosus) is a popular vegetable crop in the Northern and North-eastern states of India, cultivated for its fleshy edible tubers. Yam bean is one of the under utilized tuber crops which has been gaining important in recent years.
Major tuber crops
Aroids include taro (Colocasia), tannia (Xanthosoma) and elephant foot yam (Amorphophallus).These crops are used as vegetables and are believed to have originated in South East Asia.
Taro is a perennial cormous plant with large, heart- shaped, leaves, which are variable in size and color, arising from an underground corn. Around the central corn considerable numbers of edible cormels or lateral tubers are present. Most of them are do not set seeds in nature, but recently some genetic stocks are improved.
This is a native of Tropical America. The distinguishing feature of the plant from Taro is its sagitate or hastate leaves. Because of its resemblance of Colocassia it is called ‘coco yam’. Similar to taro this group, also does not set seed in nature.
Elephant –foot yam
This is a robust herbaceous plant, with an erect solitary pseudo-stem of 1-2.5 m in height and bearing at the top & single tripartite leaf, each part of which is divided into numerous segments. The corms are large, globose and depressed. The tubers are usually dully-yellow or brownish-yellow in colour. The crop is believed to be a native of South-East Asia.
The sweet potato originated in Central America or North-Western South America. Its entry into cultivation probably occurred about 3000 B.C. Sweet potato, Ipomea batatas (L) Lam; is a dicotyledonous plant belonging to the family Convolvulaceae. Yields of sweet potato varies with cultivate, location and production practices.
Cassava, variously called tapioca, yuca etc is a major starchy root of the tropics grown in more than 80 countries of the tropics. Botanically, the cassava plant is Manihot esculenta crantz and belongs to the natural order Euphorbiaceace.
It is a native of tropical America from where it diffused to other countries. Cassava is the 7th largest producer of staple food in the World. It ranks 3rd (7.8%) in the value of production in the tropics as compared to sugarcane (39.4%) and rice (31.2%). It is consumed in various forms as a primary and supplementary food.
Yam plants are members of the genus Dioscorea and produce edible tubers, bulbils, or rhizomes that are of considerable economic importance. They are monocots belonging to the family Dioscoreacea within the order Dioscoreales (Ayensu, 1972). Yams are the staple food stuff for millions in many tropical and subtropical countries and are a secondary food for millions more (Onwueme, 1978). Their large-scale cultivation is restricted to three main areas. West Africa, South- East Asia including adjacent parts of China, Japan and Oceania, and the Caribbean. In addition, a number of wild species have found wide spread use as a
source of the steroidal sapogenin, diosgenin, which is the precursor in the commercial synthesis of sex hormones and corticosteroids (Coursey, 1967a). These are often referred to as the medicinal yams.
Family and Genus Characters
Stem : climbing, branched, rarely short erect
Leaves : entire lobed or digitately 3-5 foliate , costate and reticular Petiole : often angular and twisted at the base.
Flowers : small or minute, pancicled racemose or spicate rarely bisexual.
Perianth : superior, 6-cleft
Male flowers : tubular, or urceolate, lobes short, spreading
Stamens : inserted at the base of the perianth, or on its lobes 3,6 or 3
perfect and 3 staminodes, flaments curved or recurved, anthers small, globose, oblong or with the cells on branches of the filament.
Pistillode : various or none
Perianth : like the male flower but smaller
Staminodes : 3, 6 or 0
Ovary : 3-quetrous, 3- celled
Styles : 3, very short
Stigmas : entire or 2- fid recurved
Ovules : 2 superposed in each cell, pendulous, anatropous, or
Fruit : a berry or 3 valved capsule
Seeds : flat or globose , embryo small , included in the hard
b. Dioscorea Linn
- Climbing herbs
Flowers : unisexual, rarely abnormally bisexual
Male flower : Perianth with six short lobes
Stamens : 6 or 3 atternating with staminodes
Pistillode : thick & flesh or zero
Female flower : perianth with six free small segments
Staminodes : 6, 3 or 0
Ovary : inferior, 3- quetrous, 3- celled
Styles : 3, very short
Fruits : a loculicidal , flatened, 3- winged capsule
Seeds : always 2 in each cell, compressed, with a large membraneous wing, albumen compressed, fleshy or hard, bilaminated, embryo between the blades, cotyledons sub orbicular.
History of the Crop
The family Dioscoreaceae is a distinct taxonomic grouping. Burkill placed 6 genera with about 650 species in the family. But in 1959, the three genera with hermaphroditic flowers were removed to other families. Most retain the six genera and place the family in its own order, the Dioscoreales (Ayensu, 1972).
According to Coursey (1967), members of the family were present and well diversified in all parts of the Southern world at the end of the Cretaceous. After this period, divergent evolution occurred in the Old and New Worlds leading to separate sections of the genus for the two hemispheres. No member of one section is found in the other. At a later time, during the Mioscene, the ancentral groups in Asia and Africa separated.
It is believed that the ancestral members of the genus were small herbaceous plants that evolved annual twinning stems and subterranean perennial rhizomatous stems. The direction of rotation of the twinning stems is either dextral or sinistral (right or left- handed) and is a taxonomic feature, i.e., all members of the section Enantiophyllum twine to the right, those of other sections , e.g Lasiophyton, Combilium, and Macrogynodium to the left . The evolution of climbing stems permitted access to better light; their annual nature coupled with perennial rhizomes or stem tubers allowed the plants to withstand drought and seasonal changes. The tubers originated by contraction of the rhizomes.
The tubers are of stem origin in all species and replaced annually. They shrink during the vegetative period of growth and new ones form and fill during the latter part of the growing season. In some species, the axillary bud or buds can develop into aerial tubers or bulbils and in one species, D. bulbifera, the bulbils provide the edible yams.
Coursey (1976b) reported that the domestication of the yams in Asia, Africa and Tropical America occurred separately, involving completely different species. In South East Asia, D. alata and D. esculenta were derived from the Indian center of origin.
D. alata then had a subsidiary distribution from the Indo-Malaysian center a region more to the southeast. From this location, D. hispida, D. pentaphylla and D.bulbifera are also originated.
Table .1. Major Edible Yam Species
Species Common name Location
D. alata L. Greater yam Orgi. Southeast Asia
D. rotundata White Guinea yam West Africa
D. cayenensis Yellow Guinea yam West Africa
D. nummularia Indonesia, Oceania
Chinese yam China, Korea, Taiwan, Japan
D. dumetorium African bitter or cluster yam West Africa
D. hispida Asiatic bitter yam India, Southern China, New Guinea
D. esculenta Lesser yam Tropics, esp. Asia & the Pacific
D. bulbifera Potato or aerial yam Orgi. Asia & Africa cult: tropics
D. trifida Cush-cush yam Orgi. New World (northern South America) Cult. Caribbean
Utilization and Economic Importance of Yams
The tuber is the main economically utilized part of the yam plant. The chemical composition of the tuber varies with species and cultivar; even within the same cultivar, it may vary depending on the environmental conditions under which the tuber was produced.
The largest component of the fresh tuber is water (65-75%). Carbohydrates are the major dry matter content of yams. Most of this carbohydrate is starch (15-25%). Starch itself is amylopectin and exists in the cells as starch grains. In D. bulbifera, triaagular in shape and in D. esculenta, they are small & angular in shape.
Sugars present only in minute quantities. For most species, they account for less than 1% of the fresh weight, but in D. esculenta which is sweet, the % of sugar may be as high as 2-4% on a fresh weight basis. The sugar present mostly is sucrose, but small traces of glucose and fructose may also present.
Protein content of yams is rather low, ranging from 1-2% of the fresh weight. Mucilage which exudes when yam tuber surface is cut are mostly glycoprotein.
Vitamins and minerals (ash-0.7-2%) are also minor components of yam tuber. Significant amount of vitamin C are present, with values in the range 6- 10 mg/100g of tuber tissue. Traces of vitamin A & B are present. Ca, Fe, P are among the components of the mineral fraction of the tuber.
Some yams may contain small traces of polyphenolic compounds. Some contain alkaloids (e.g. dioscorin) and steroid derivatives (e.g. diosgenin).
As Herbal Medicine
Tubers of Dioscorea are used throughout the world as food & herbal medicine. These true yams are used to treat inflammation, joint pain, diabetes, infections and dysmenorrhea.
The pharmacologically active components of Dioscorea include:
(1) Diosgenin - steroidal saponin – used to make steroid hormones.
(2) Dioscin - diosgenin with sugar attached form
Diosgenin - functions as a phytoestrogen
- lowers plasma- cholesterol level
- Reduce blood- sugar level
- Decrease inflammation
- Protection against bacterial infection and cancer
Yams for Food
By far the largest proportion of yams produced annually is marketed as the fresh tuber. Only a very small fraction goes to market in processed forms. Boiled yam is one of the simplest and commonest forms in which yam is consumed. Pounded yam is perhaps the most traditional form in which yam is eaten in West Africa. It is prepared from boiled yam. Yam flour and yam flakes are also produced from yams. Yam chips, for snacks are also a relatively new form of processed yam.
Other uses of yams
The main sapogenin present in yams is diosgenin, which serves as a starting point for the pharmaceutical manufacture of several corticosteroid drugs. The alkaloids present in yams include dioscorine in D.hispida, and dihydrodioscorine in D.dumentorum. Both the substances are nerve poisons, and the species that contain them are therefore used as sources of poisons for hunting, fishing & pharmaceutical purposes. They are soluble in water, so that tubers containing high amount of alkaloids can be detoxified by prolonged soaking in water.
The largest acreage and the greatest amount of yam production is in West Africa. This region accounts for over 95% of total world acreage and production of yam.
West Indies is the second most important yam- producing region in the world. Jamaica, the Dominican Republic, Haiti & Puerto Rico are the major countries of production there.
The third important region of yam production is East Africa, where Sudan and Tanzania are the major producers. D. roundata is the most important species in West Africa, while D. alata is the most important in the West- Indies. It seems that, on a world –wide basis, the greatest acreage and production of yam is of D. roundata. D. alata is next in importance, followed by D. cayenensis and D. esculenta in that order.
Most of the yam produced in various parts of the World is consumed within the country of production. Very little enters into the international trade. The
economic importance of yam, therefore, lies in its utility as a carbohydrate, food for producing region rather than in any ability to earn foreign exchange.
Ethno- Agricultural Importance of Yams
For several decades, before the introduction of maize and cassava, yam was probably the main sustenance of the peoples of the West African yam zone. During that time considerable importance must have been attached to the success or failure of the yam crop. ‘Yam festival’ was celebrated by the people. A date is fixed for the festival, and before that date, no farmer may harvest or consume the new crop of yams. On the appointed day, each farmer harvests several yams from his farm, uses part of the harvest to prepare a feast, while the remainder may be used to pay homage to his elders or friends. Now a day’s ritualism developed around the production and utilization of yams.
Propagation of Yams
Yam plant can be propogated by tuber, bulbil, seed, and vine cuttings or by tissue culture. Propogation by tuber is by far the most common and is commercially the most important. In nature tubers serve as a major organ for perennation, enabling the plant to survive adverse seasons and to regrow the following year. Partly, for this reason, many of the wild relatives of yams and even some cultivated types, posses extensive thorns which surrounds the tuber and protect it from the foraging activities of various animals.
Propagation by Seeds
As a general rule, plants that have propagated themselves vegetatively for long periods of evolutionary times tend to lose their ability for efficient sexual reproduction of the major species of yam, D. dumetorum, D. bulbifera and D. trifida flower and produce seeds regularly. But some of the species have irregular
flowering. So propagation by seed is difficult in such species. Sometimes the seed is dormant (for 3-4 months) after maturity and can not be made of germinate immediately.
The propagation of yam by seeds, even though difficult, is nonetheless significant for several reasons. Firstly, yam propagation by seed is an extremely valuable tool in attempts to improve the crop by breeding. The sexual reproductive process which produces the seeds also results in hybridization.
This in turn results in large production of a number of genotypes which can form the basis of selection.
Secondly, it is known that many virus and nematode diseases of yam can be transmitted through the tuber but not seeds. Also all the tuber material that is now used for planting would be available for consumption & the inedible seed would be planted.
Propagation by vine Cuttings
Although non-edible Dioscorea have long been propagated by vine cuttings, the ability of vine cuttings of edible yam plants to root was 1st reported by Njoku (1963) for D. alata, D. rotundata and D. dumetorum. Since then, this method of yam propagation has become more common. This method essentially involves taking a piece of the yam vine, causing it to root in water or a moist substratum and growing the established plant to maturity.
To ensure the greatest rapidity of rooting, the cutting must contain a single node preferably at its lower end.
The propagation of yams by vine cuttings is a very useful technique for rapid multiplication of desirable clonal material. It is possible to produce hundreds of separate rooted plants in a very short time starting from one single plant. Propagation by vine cuttings is an effective means of producing plants that would be free of certain diseases such as roots and nematodes.
Propagation by bulbils
Yams can be propagated by their bulbils. In some such as D. bulbifera, bulbils formation occurs readily, and the crop is often grown for the edible bulbils. In others, such as D. alata, bulbil formation occurs less readily, but could be enhanced by treatments which obstruct the translocation of food material to the underground tuber.
Yam propagation by bulbils bears several distinct similarities to propagation by tuber. The bulbil exhibits dormancy, there is a head region from which sprouting occurs preferentially; and the yield is influenced by the size of the bulbil material used for planting. The larger the bulbil or bulbil - piece used for planting the more robust the plant that results. Plants grown from bulbils reportedly produce small tubers (Coursey, 1967a).
Propagation by Tissue Culture
Tissue culture techniques are a fairly recent development in plant physiology and the propagation of yam by tissue culture is even more recent. Yam propagation by tissue culture offers the ultimate in clonal multiplication. From a single tuber blocker nodal section it is theoretically possible to produce millions of little yam plants within a single year. It is, therefore, a method that offers promise when rapid multiplication of a single desirable plant is required.
Advantages of Micropropagation
In vitro micropropagation techniques are now often preferred to conventional practices of asexual propagation because of the following advantages.
1. A small amount of plant tissue is needed as the initial explant for regeneration of clonal plants in one year. In comparison it would take years to propagate an equal number of plants by conventional methods.
2. Micropropagation helps in bulking up rapidly new cultivates of important trees that would otherwise take many years to bulk up by conventional methods.
3. The in vitro techniques provide a method for speedy international exchange of plant materials.
4. The in vitro stocks can be quickly proliferated at any time of the year. Also, it provides year round nursery for ornamental, fruit and tree species.
5. Production of disease free plants: through meristem tip culture virus- free plants were obtained in potato, sweet potato, yams, gladiolus etc.
6. Seed production: For seed production in some of the crops, major limiting factor is the high degree of genetic conservation required. In such cases micropropagation (axillary bud proliferation method) can be used.
7. Germplasm storage: Preservation of germplasm can be done using tissue culture.
Diseases and pests of yam & their control
Insect pests are a serious problem in the production of yams. The yam beetle (Hereroligus spp) is a major pest of yams in West Africa. Yam beetles rarely kill the yam plant, but they do considerable damage to the tuber that can be harvested from it. Their feeding activity leaves hemispherical holes on the body of the tuber. The main control measure now for yam beetles is to dust the set with an
insecticidal dust just before it is planted. Aldrin, BHC or gammatin have been found effective.
Chrysomelid beetles do most of their damage to the young yam. Foliage sprays of insecticides are used to control infestations of chrysomelid beetle larvae. Sevin 85, DDT, or Agrocide 3 has been effective. Other insects which cause damage to yam are Sciarus flies, scales and mealy bugs, weevils, termites, grass hoppers, and aphids.
The yam nematode, Scutellonea bradys, the root not nematode (Meloidogyne species), the root-lesion nematode, pratylenchus species , are the more common nematodes which are required for their control.
Tuber rots, leaf spots, leaf blight, rusts, crown gall etc are the major fungal diseases of yams, whether in storage, the planted sett or the growing tuber in the field. Fungal diseases of yams cause considerable losses in D.alata in West Africa.
Virus infections are associated with two major types of symptom in yam: leaf mosaic and leaf shoe string. In leaf mosaic, the leaves are mottled in appearance with yellow blotches and some degree of vein clearing. The plant may look normal. In the shoe string symptom, the entire plant or parts of it are usually dwarfed and many of the leaves are lance orate and variously curved. No effective control measures for these viral diseases have yet been devised. Mild heat treatment of setts, before planting may effectively control tuber borne viruses.
Bacteria contribute to the rotting of yam tubers. Serratia have been implicated (Okafor, 1966) but other genera are probably involved as well.
Rodents are other major pests of yams.
II REVIEW OF LITERATURE
The genus Dioscorea consists of about 600 spp belonging to the family Dioscoreaceae under monocotyledons. Apart from the edible spp. the genus also includes wild, poisonous and medicinal spp. Dioscoreaceae was formerly classified under Liliales (Burkill, 1960: Cousey, 1967 a) but has now been separated and grouped together with Trichopodaceae and Roxburgiaceae to form the order Dioscoreales (Ayensu, 1972; Ayensu & Coursey, 1972)
Yams constitute a group of Dioscorea spp. cultivated widely in the tropics for their edible underground tubers. Yams are considered as crops of ancient origin which were domesticated before 500 B.C. (Alexander and Coursey, 1969). The species are divided as old and new world groups based on their origin, distribution and evolutionary history. These groups were separated at the end of cretaceous by the formation of Atlantic Ocean, which led to their further divergent evolution (Burkill, 1960). Among the old world group there are Asian and African species. And the New World group represents the Mexican and Central American species.
The edible yams consist of about 50 spp. including those used as famine foods and many marginally cultivated spp. (Cousey, 1967; Martin and Degras, 1978). About 10 of them are considered as food yams of which three, viz. greater yam (D. alata), lesser yam (D. esculenta) and white yam (D. rotundata) form the important yams of the tropics. The former two is Asiatic in origin and the latter is African.
Prain and Burkill (1936-38) reported 90 spp. under the genus Dioscoreaceae from the subcontinent. Fischer (1928) reported 14 species of the genus are considered to be useful to mankind as food (Harlan, 1975) or as medicinal and poisonous plants. Only about 6 major species are cultivated for food purpose and these are known as true yams.
The genus Dioscorea includes over 600 species, which are distributed all over the humid intertropical zone. In India following species are present widely.
2.1 The cultivated species are
a. D. alata
b. D. esculenta
2.2 The wild species are
a. D. bulbifera
o. D.wighti etc
The various species of edible yams will now be discussed with respect to their characteristics, origin and distribution, extent of cultivation, and cultivars within the species.
Some of the major edible yams are D. alata, D. esculenta, D. rotundata, D. cayenesis etc.
i. D. alata L
It is the most widely distributed species of yam. It is also known as water yam or greater yam. Tuber shape is extremely variable and complicated by tuber branching but mostly cylindrical, brown to black in colour. Tuber flesh is white and watery in texture. Some cultivars also produce aerial tubes in the leaf axils which are edible. D. alata originated in south-east Asia, probably in Burma. Several cultivates of D. alata exist, being distinguished by tuber and shoot characteristics (Onwueme & Charles, 1994).
ii. D. esculenta (Lour) Burk.
It is also called lesser yam or Chinese yam or Asiatic yam. It has a stem which twins to the left. The tubers are small and characteristically borne in clusters by each plant, unlike most other yams which usually produce only one or two large tubers per plant. Each plant of D. esculenta may produce 5-20 tubers. Each tuber is almost cylindrical, with rounded ends. The tuber flesh is white and less fibrous than that of most other yams. D. esculenta is originated in Indo- China, and has been cultivated in China since at least the 2nd century A.D. Its cultivation has, now spread throughout the tropics. Mainly two types of D. esculenta are present.
1. Wild types- have vigorous foliage, long stolons, fibrous tuber flesh, prickly climbers and roots.
2. Cultivated types- flowering is rare.
(Onwueme & Charles, 1994).
iii. D. rotundata Poir.
It is grown on a greater hectarage than any other yam species in the world. It is also known as white yam or white guinea yam. The stem twines to the right and will usually grow to a length of several meters. The tuber is more or less cylindrical in shape. D.rotundata is native to West Africa. It is introduced to India in 1976 through IITA Nigeria in the form of seeds and now it is greatly established.
iv. D. cayenensis Lam.
It has many similarities with D. routundata. It is also known as yellow yam or yellow guinea yam. It was introduced from West Africa to West Indies during 16th Century. Today its cultivation is mainly confined to these two areas, and no significant amounts are grown in Asia. Tuber flesh is usually yellow due to the presence of carotenoids; hence it is called ‘yellow yam’.
D. bulbifera L (aerial yam) is characterized by the production of large numbers of bulbils (aerial tubers) on each plant. Each bulbil originates in the axils of a leaf. Dioscorea bulbifera is the only edible yam species i.e native to both Asia and Africa. Wild forms of it can be found on both continents. The Asiatic varieties produce bulbils that are generally less angular, more spherical, and less toxic than the African ones. The cultivation of the species is most prominent in the West Indies and the South Pacific Islands.
(I .C Onwueme, 1978).
D. tomentosa Koenig ex Spreng is a medicinal yam whose thread like fibro-vascular bundles cause the yam to be known as the ‘thread yam’ (Prain and Burkill, 1936).
D. glabra Roxb. is also a medicinal yam. It is a wild species not found universal distribution through the western and the eastern Indo- Chinese phyto-geographical sub regions, and also extensions over the borders into the Indian and
Himalayan sub regions on the west. The species is glabrous in nature (Prain and Burkill, 1938).
2.3 Dioscorea bulbifera L
It is a wild species native to both Asia & Africa. It is also known as air potato, erachikachil, urulakizhangu kachil etc.
Kingdom - Plantae – Plants
Sub kingdom - Treacheobionata – Vascular plants
Super division Spermatophyta- Seed plants
Division - Magnoliophyta – flowering plants
Class - Lilliopsida- Monocotyledons
Sub class - Lilidae
Order - Liliales
Family - Dioscoreaceae- Yam family
Genus - Dioscorea L- Yam
Species - D. bulbifera – bulbil producing
History and Origin
D. bulbifera is the only edible yam species native to both Asia & Africa. The African varieties are so distinct from the Asian that their distribution must have taken place in prehistoric times. Perhaps the species had two centers of diversity, one in Indo-China , Malaya or Indonesia, and the other in Africa. The major difference of shape is that the Asian forms are more or less spherical and the African forms are angular.
Probably D. bulbifera can be found in every hot, humid, tropical region. Burkill speaks of D. bulbifera as having extended to the most remote islands of the pacific. Wild forms, usually bitter and often poisonous are the most common.
D. bulbifera is not native to the Western Hemisphere. Nevertheless, it is so widespread that it is noted in most floras of the tropical countries of Central and South America.
D.bulbifera is a glabrous vine that climbs by twining to height of 12 m or more. The stems range from thin to thick and twist to the left in twining. The leaves often quite large, are alternate and unusually orbicular, but with well-developed acuminate tails. They are cordate at the base. The leaves are usually glabrous, but in some varieties they are covered with a bluish bloom. The petiole which is enlarged at the base has ear-like project and implimentationions (auricles) that encircle the stem.
Only small seedlings have true roots, and these are short-lived. The principal adventitious roots arise from the crown, the region of the stem immediately above the tuber. Finer roots arise from the surface of the tuber itself in some, but not all varieties.
The small flowers are frequently not seen during summer. They are sessile and appressed to the peduncle in long racemes produced in the axils of the leaves. The male and female flowers superficially appear alike, but the female is easily recognized from its inferior ovary. The perianth ranges from green to white to slightly pink. The flows produce a pleasant odor, which attracts bees and other insects.
The seed capsules are trilocular, 2-5 cm long and rise vertically from the racemes. The seed is surrounded by a membranous falcate wing, which is hooked at its attachment to the placenta.
In common with many yam species, D. bulbifera produces both underground and aerial tubers (bulbils). Bulbils are also edible. Bulbils begin to develop in the axil between leaf and stem about the time that the leaf itself begins to unfold, and they grow rapidly. Aerial tubers eventually fall from the vine. The time of fall depends on the variety. In some varieties tubers of all sizes fall, but in the better varieties, the tubers reach a suitable size and maturity before falling.
The axil of each leaf contains two bud primordia and a bulbil primordium. It is the bulbil primordium that proliferates to give rise to shoots, roots, and tubers. When vine cuttings of D.bulbifera are palnted. The underground tuber is small in the Asian and African varieties which are often grown for the bulbils. D. bulbifera is characterized by their large production of bulbils.
The flesh of the aerials & under ground tubers is crisp, fine grained, and almost free of fibers. Color varies considerably among varieties. Many varieties contain anthocyanins, which in small quantities and combined with yellow pigments produce pinkish or grayish flesh. A small amount of chlorophyll is also present in some species.
Two important studies have been made of D. bulbifera varieties. The African varieties have been treated in depth by Chevalier and the Asian species by Prain and Burkill. Varieties are distinguished by their following characteristics: size and shape of aerial tuber, size and shape of under ground tube, size and shape of leaves , diameter of mature stem, color of flesh, bitterness of cooked flesh , prominence of lenticels on aerial tuber & minor characteristics of leaves. Some of the African Asian varieties are: senegambica, sylvestris, anthropophagorum, violacea, longipetiolata, heterophylla, sativa etc.
Analysis of tubers give the following information
Albuminoids - 7.36-13.31
D. bulbifera tubers contain high amount of starch. But immature tubers contain fewer amounts. Amylase content of starch is moderately low. D. bulbifera starch has a low viscosity and high gelatinization temperature.
The protein content is very valuable. But the quality of the protein is poor. Protein also lacks tyrosine and lysine.
The yellow pigments of D.bulbifera unfortunately have no sufficient nutritional value. The carotenoids are entirely xanthophylls and their esters.
Poisonous or Obnoxious Contents
The tubers and bulbils of some varieties of D.bulbifera are poisonous. On the Island of Java the aerial tubers are used to make fish poisons. Chevalier distinguished his varieties on the basis of its poisonous content.
One of the poisonous substances of D.bulbifera is apparently dioscorine, an alkaloid found in many poisonous yams and sometimes used as heart stimulant. Saponin content was also identified in some varieties. The tubers are detoxified by certain treatments such as prolonged or repeated heating, sleeping the tuber
slices in running water, and pound the tubers with lime or sand and then roast them slowly.
The aerial and underground tubers have been used many ways in folk medicine. In India the bulbils have been used externally for sores and internally for hemorrhoids. Among the Santhals of central India a paste from the tuber is used for swellings and as a cure for snakebite. In Jamaica, the tuber is used for the treatment of scorpion stings and ulcers. In Africa extracts are used in toddy to stimulate excessive drinking. Dried and pounded tubers are also used in piles, dysentery and syphilis.
Tubers are used for the preparation of starch in Japan. Tubers are used in Kashmir for washing wool and as fish bait.
Pests and diseases
Some varieties of D. bulbifera are some what susceptible to the leaf-spot diseases (especially cercospora) seen in other species. Usually such diseases seen only late in the season, when the foliage is beginning to die back normally. Stem rot sometimes occurs, but it is not a serious condition. Premature drop of partially developed bulbils is frequent and may be associated with minor fungal infections during rainy weather. The underground tubers are sometimes attacked by nematodes and beetles, but they appear to be more resistant than tubers of other species.
2.4 Dioscorea glabra Koenig ex Spreng
It is also a wild species. D. glabra of general, if not universal, distribution through the Western and the eastern Indo-Chinese phyto geographical sub regions,
and have extensions over the borders into the Indian and Himalayas sub regions on the west, the Malasian on the South.
Kingdom - Plantae
Subkingdom - Tracheobionata
Super division -Sprematophyta
Division - Magnoliophyta
Class - Lilliopsida
Subclass - Lilidae
Order - liliale
Family - Dioscoreaceae
Genus - Dioscorea
Species - D. glabra
Morphology of the species
One or two or more, produced as swellings on the ends of long stalks descending from a woody knot of tissue at the surface of the soil; on this woody knot leaf-scales may at times be found. Each tuber attain 50 cm in length with a diameter of about 4 cm, the stalk nearly as long as or sometimes longer than the swollen part of the tuber: flesh white, edible and delicate: skin earth colored, with a few rootlets.
Climbing to a height of about 8m, those of the first and second year’s plants life unarmed, but after that in the lowest 10cm. Close to the ground freely armed with abundant recurved prickles, above unarmed, glabrous, smooth, livid green.
Not observed and probably never formed.
Rarely exactly ovate, commonly elliptic- ovate & in var. longifolia long elliptic- ovate, rounded or slightly cordate at the base: hastate leaves sometimes found and probably when found only due to abnormal condition such as removal of overhead canopy; the blades of typical leaves very thin in textures and in wilting in rolling from the margins; those of var. grisea some what firmer, 5-nerved; petiole usually 4-5 cm long.
In spikes upon long leafless branches or branch- endings, or more rarely in fascicles in axils; the leafless branches may attain 70 cm in length and carry upwards of 15 false whorls of spikes. Spike up to 4 cm long, its axis angled, glabrous, its flowers to 25 in number usually so closely packed that they touch each other: buds sub globose above their broad base; bracts broadly ovate, shortly acuminate. Hardly 1mm long. Bracteole similar but smaller. Sepals from a broad base ovate, obtuse about 1mm long, glabrous .Petals a little shorter, obovate, rounded above. Stamens -6, the anthers in length equaling the filaments. Gynoecium a 3- pointed cone.
In long curved solitary spikes which attain 40 cm in length and carry upwards of 50 flowers, axis glabrous, angled: bracts ovate, acuminate about 1mm long, bracteoles similar smaller. Sepals triangular, ovate, thick about 1mm long. Petals similar, smaller. Staminodes minute. Stigma as three pairs of sickle-like hooks. Capsules pale green until they reach maturity, when they turn yellowish and then tawny: the stipe 4mm long, widening upwards to a diameter of 3mm. Seeds with a broad smoky wing all round, conform to the locules.
2.5 D. tomentosa Roxb.
Kingdom - Plantae
Sub kingdom - Trachebionata
Super division -Sprematophyta
Class - Lilliopsida
Subclass - Lilidae
Order - Liliale
Family - Dioscoreaceae
Genus - Dioscorea
Species - D. tomentosa
Several, descending in a bunch from the knot of tissue of the surface of the soil and growing to a depth of 1-2m, nearly cylindrical throughout, and sometimes branched, with a hard brown roughly granular skin and soft white flesh which is transfused by strong vascular fibers; short roots arise from the surface in the upper part and long feeding roots are developed in the surface soil above them.
Usually solitary and not branching for a little distance above the base, in total length perhaps as much as 20 m, generally sparingly prickly, when young densely tomentose with white hair, when old showing slight ridges near the base, at the most 5 mm thick.
Bulbils- not seen
Variable, having all possible number of leaflets up to 5, those low down being richest in leaflets and the simple leaves being towards the apices of the branches. Common petiole up to 12 cm long: median leaflets elliptic-ovate, abruptly acuminate up to 15 cm, long by 5 cm in width or narrower, being very variable in width .Petioles- 2-6 mm long. Simple leaves broadly cordate or ovate, 5-7 nerved.
-in spile –like racemes which are grouped together into a paniculate inflorescence and this inflorescence may attain as much as 30 cm in length: axis of the spike- like racemes about 3-4 cm long, densely pubescent: the flowers upon it in number about 20-30. Pedicels short, buds subglobose, bracts ovate or sometimes almost circular, bracteoles similar, smaller. Petals similar, very slightly smaller than the sepals and less pubescent. Stamens 3, rather shorter than the
sepals from the base of which they arise: anthers as long as the filaments: staminodes 3 about equaling the stamens in length or a little longer, fish- tailed at the apex. Gynoecium a small three- lobed wart.
- in long simple dependent spikes, which are solitary or two together in the leaf-axils. Axis up to 35 cm, long, pubescent, with the flowers facing towards until anthesis, when they gradually become reflected: bracts ovate, acuminate ; 2mm long. Sepals thick, ovate. Petals thinner, smaller, ovate, subacute. Staminodes 6, small. Stigma of 3 pairs of short rays. Ovary about 6 mm. long at flowering. Capsules up to 25 mm. long the wings rounded at both ends, but broadening a little upwards, in all 24 mm long by 10 mm in width, retaining the pubescenece till maturity, dehiscing to the base both loculicidally and along the placentas. Seeds winged towards the base of loculus, usually 15-20 mm long.
Thread like fibrovascular bundles cause the yam to be known as the ‘thread yam’.
2.6 History of Tissue Culture
The culture of plant cells or plant tissues in a synthetic culture medium under controlled aseptic conditions is known as tissue culture. It is also called in vitro culture. The culture medium provides all the minerals and growth hormones necessary for the growing cells.
All plantlet regenerated from a plant material are identical and similar in the metabolic activities and are known as clones. The method of raising the clones is termed micro propagation or in vitro propagation via plant tissue culture.
In 1902, a German Botanist Gottlieb Haberlandt (in Berlin) developed the concept of culture of isolated cells of Tradescantia in artificial condition. Though this experiment failed to induce the cells to divide, he did not succeed because by
that time even auxin was not discovered. But he lent a foundation to plant physiology. He described the cultivation of mesophyll cells of Lamium purpureum and Eichhornia crassipes, epidermal cells of Ornithogalum and hair cells of Pumonaria. Cell survived for 3-4 weeks. Due to this endeavor, Haberlandt is regarded as the father of tissue culture.
From 1902-1930 attempts were made for organ culture. Hanning (1904) isolated embryos of some crucifers and successfully grew on mineral salts and sugar solutions. Simon (1908) successfully regenerated a bulky callus, buds, roots from a poplar trees on the surface of medium containing IAA which proliferated cell division.
Kotte (1922), a student of Haberlandt in Germany, and, independently Robbins (1922) were successful in the establishment of excised plant root tips in vitro. However, in 1934, the pioneering work of growing excised roots of tomato in vitro for periods of time without theoretical limits was demonstrated by White (1934). Initially, White used a medium containing organic salts, yeast extract and sucrose, but later yeast extract was replaced by three B-vitamins viz pyridoxine, thiamine and nicotinic acid.
Until the early 1930s, R.P. White (USA), Gautheret (France) and Nobercourt (France) independently cultured tissues excised from several plants on the defined nutrient media for a long period. Gautheret (1939) cultured cambium tissue of carrot on Kop’s solution supplemented with other chemicals in trace amount. White (1939) cultured tobacco tumor tissue from tobacco hybrid.
During 1940-1970 suitable nutrient media were developed for culture of plant cells, tissue, protoplasts, anthers, root tips and embryos.
In the 1950 s several important achievements were made in the field of plant physiology. The foundation of commercial plant tissue culture was laid in
1960 with the discovery of G.M. Morel for a million fold increase in clonal multiplication of an orchid, cymbidium.
Miller et al., 1955 identified kinetin. Later on other cytokinins such as zeatin, and isopentyl adenine were discovered. Skoog and Miller (1957) advanced the hypothesis of organogenesis in cultured callus by varying the ratio of auxin and cytokinin in the growth medium.
In 1953, Muir reported that if fragments of callus of Tagetes erecta and N. tabaccum are transferred to liquid culture medium and the medium is agitated on a reciprocal shaker, then the callus fragments break up to give a suspension of single cells and cell aggregates (Muir, 1953). He further developed a nurse culture technique. Bergman (1960) developed another technique for doing large numbers of single cells.
Cell wall creates a barrier in plant protoplast culture. In1960s, the role of enzymes e.g. cellulose and pectinase in dissolution of cell wall in buffer solution at suitable pH, and isolation and culture of protoplast was developed (Cocking, 1960).
Guha and Maheswari (1966) developed techniques for the production of vast numbers of embryos from cultures of pollens and sporogenous tissues of anther. Nitsch (1974) gave methods to double the chromosome number in microspores of Nicotiana and Datura.
In India, work on tissue culture was started during mid 1950s at the Dept of Botany (University of Delhi) by Panchanan Maheswari who is regarded as father of embryology in India.
2.7 Applications of Tissue culture Technology
Tissue culture is used to maintain and grow plant tissues (callus, cells, protoplast) and organs (stems, roots, embryos) in aseptic (or in vitro) culture.
Techniques used to regenerate plant through tissue culture.
Seedling formation → through 1. Seed culture
2. Embryo rescue
3. Ovule culture
4. Ovary culture
Plantlet formation → 1. Axillary shoot formation
a. Meristem culture
b. Shoot culture
i. Nodal cultures
Callus formation → 1. Callus cultures
2. Callus suspension cultures
3. Protoplast cultures
Plantlet formation → Adventitious shoot formation
1. Diploid plant regeneration
(Leafpieces,bulbscales,petioles,roots, stem, internodes)
2. Haploid plant regeneration [(Pollen mother cells) Anther)]
Somaticembryoformation→ 1. adventitioussomatic embryogenesis
2. Induced somatic embryogenesis
2.8 Plantlet regeneration by micropropagation
Axillary shoot formation
Meristem culture/ shoot tip culture
Shoot tips of plants are used in micropropagation (for virus elimination). Meristem-tip culture has been successful with many important herbaceous crops. It is effectively worked in carnation, chrysanthemum, orchid, potato, cassava, sweet potato etc. The tip of axial organs of a plant shows potential for unlimited growth because of the presence of apical meristems constantly undergoing cell division and cell differentiation. Stem tip culture of citrus plants from bud meristems and small apices failed to grow in vitro but entire buds & shoot apices could be maintained in culture for several months. Shoot apices of Jasmium grown on a nutrient agar medium developed callus in profusion with potential for regeneration of shoot lets but not from the calli of other parts.
A basal medium of MS or Gamborg B5 containing a combination of an auxin (1mg/l), a cytokinin (2mg/l) and GA3 (0.1mg/l) plus sucrose is frequently used for a variety of shoot tips of herbaceous species for successful growth to a shoot. (S. Narayanaswamy, 1994). Shoot tip or axillary bud culture is a unique method of production of genetically identical plants for clonal propagation for commercial purposes.
Organ Culture of the cole crops (plants within the genus Brassica) representing cabbage, mustard, cauliflower, turnip etc has been carried out for the purposes of regeneration, from meristem tip (Walkey et al., 1980), hypocotyls and cotyledon (Hui and Zee, 1978), axiallary bud (Kuo and Tsay, 1977), and ovary (Inomata, 1976). Vegetative propagation has been successful through meristem culture in Brassica oleracea var.gemmifera (Clare and Collin, 1974).
The cactus, Opuntia polycantha is a species exemplified by direct organogenesis from axillary meristems influenced by phytohormone supplements (Mauseth and Halperin, 1975). Application of BAP (5-10 mg/l) to region of the areoles resulted in the regeneration of leaf green shoots whereas fibrous spines devoid of vascular elements were formed with added GA3 (20-100 mg/l). Exogenous NAA (5-50 mg/l) caused the development of root at the base of the presumptive bud primordia of aereoles.
Shoot apical meristem culture as a technique has been of widespread practical application to Horticulture and Agriculture in the large scale – production of plantlets of economic value and in the recovery of plants that are virus free. Auxin in low concentration in the medium stimulates the development of axillary adventitious shoots directly but at higher levels, causes cell proliferation of the explants forming a callus mass. Seedling parts and juvenile tissues (from trees) responding better than mature parts in hormone response. The conservation of in vitro material permits the creation of the disease-free gene banks (Boxus and Druart , 1980).
It was not until the last decade that tissue culture technology was applied to root and tuber crops (Kartha, 1981) and especially to cassava. Meristem tip culture of cassva became popular during the last decade and research was oriented towards elimination of virus disease for the recovery pf healthy cones (Kartha, 1981). On culture in M.S medium containing 0.05 µM NAA and increasing the concentration of BAP (2.5 to5.0 µM), the shoot tips gradually developed into dense clusters of shortened shoots comprising several nodes forming rosettes. Growth of axillary buds of rosette cultures at each node, in their turn gave rise to precocious axillary shoots and so on.
Probably the best known work on several cultivates of Ipomoea batatas apical meristem culture (10µ) is that of Mori (1971) and Liao and Chung (1979) to obtain plants free of known viruses and to shorten the time required for regeneration.
Shoot multiplication through shoot tip culture of Helianthus annus is successful in a medium containing BAP or Kinetin only but not with other cytokinins. Increasing the concentration of BAP resulted in rosette shoots with poor survival (Paterson, 1969).
Long shoots are cut into single nodes and planted vertically in the medium. Axillary buds at each node elongate and grow in length. This is repeated by again cutting into nodal segments at each subculture.
Growth of multiple axillary shoots is stimulated when nodal explants are cultured in a medium with high cytokinin/ auxin ratio as in white clover. (Bhojwani, 1981). Rootless shoots are isolated and cultured in a semi-solid medium with low sucrose concentration and one-or more auxins such as IAA, IBA or NAA for rooting of individual shootlets. In cases of plentiful formation of
multiple shoots, the internodes are shortened on the stem and the shoots present a crowded appearance rendering their separation difficult. (S. Narayanaswami, 1994).
Multiplication through stimulation of axillary branching of excised nodal parts is yet another method largely applicable towards propagation of woody trees where regeneration from calli is difficult and rare, and exhibit somaclonal variations (Larkin and Scowcroft, 1981).
Minitubers for culture
Potato plants in culture form miniature tubers at the end of small stolons extending from the lateral meristems located at each node of potato plants when treated with high levels, particularly in the darkness. These storage organs can be removed and used in the production of virus-free planting stock. Yam produces root tubers at the base of stem node cuttings. (S. Narayanaswamy, 1994).
Sterile culture of isolated young immature leaves of a number of plants has been attempted with a view to ascertaining the nutrition and growth of leaf primordia to maturity in vitro. Leaf explants of apple seedlings in vitro have shown adventitious embryogenesis (Liu Jr. 1983)
Shoots regenerated from leaves of Vitis vinifera in vitro on MS medium + 2mg/l BAP, when transferred to agar media, grew into complete plants (Stamp,J.A ,1990). The induction of adventitious buds from leaves has been reported from a few woody angiospermic genera such as Sapium .
In vitro organogenesis and somatic embryogenesis from leaf explants of Leucosceptrum canum have been reported (Amita, 1985).
Adventitious shoot formation on leaves still attached to young sugarbeet plants (Beta vulgaris) was observed when cultured with high concentrations of BAP (2000 mg/l) (Saunders and Mahoney, 1982).
The stimulatory action of BAP in inducing multiple buds on explants of stem and leaves was a striking feature in Petunia (Rao et. al., 1978), Nicotiana tabaccum (Praphudesai and Narayanaswamy, 1979) and Brassica spp (Kartha ,1974)
Explanted leaves of ornamental plants such as Begonia, Peperomia, Achimenes, Kalanchoe blossfeldiana, Saintpaulia and Streptocarpus have also shown ability to form adventitious shoots directly from leaves without the intervention of a callus phase and from petiole sections (Hartmann and Kester, 1975; Bilkey et al., 1978; Smith and Nightingale, 1979; Hussey, 1980).
Callus cultures represent clumps of unorganized parenchyma tissue formed by the vigorous proliferation by cell division from the small explants in culture, showing no polarity. Living but apparently quiscent tissue such as collenchyma, cortical parenchyma, pith cells, phloem tissue and cambia become active and rejuvenated under culture conditions. They are stimulated to undergo cell division under the influence of endogenous and added growth substances in the medium. Successful callus cultures can be established from nearly any part of the plant.
The transformation of an explant into a proliferating callus mass under culture conditions is reflected in a change in the basic pattern of the tissue by cell division. Loss of cell types, development of new cell types and quiscent cells becoming metabolically active are some examples (Gauthret, 1966). Callus induction also depends on the plant genotype, the source of origin of explant and the physiological state of the tissue culture. (Murashige, 1974; Thomas et al.,1979 Harms, 1982).
In several conifers (Picea ables, Pinus strobus and others), embryonic shoots could be regenerated via calli but rooting has been a problem (Jansson and Bronman, 1980; Minocha, 1980).
2.9 History of in vitro culture of yams
In vitro culture of yams dates back as far as 1958, at least, with the experiment by Swada et al., of the effects of sucrose and peptones on the organogenesis and growth of bulbils from leafless nodes of Dioscorea opposita. Bulbil organogenesis again in 1968 (Asahira and Nitsch, 1968) and, then, with D. bulbifera in 1970 and 1971 (Uduebo) remains the first approach of the technique, until about 1975 a widest interest begins for in vitro yam culture, applied primarily to the diosgenin producing species.
Most researchers used directly part of the entire leafed or leafless node as material.
Some researchers used
→ Leaf of D.deltoidea (Mascarenhans et al.,1976) or D.floribunda (Sinha andChaturvedi;1979)
→ Seedling hypocotyl of D. deltoidea (Grewal and Atal, 1976) →Tuber of D. deltoidea (Mascarenhas et al., 1976, Singh, 1978)
→ Apical meristem of D. deltoidea (Grewal et al., 1977).
Mapes and Lirata (1970) could not obtain any proliferation from D. spp internode.
As in many cases with other plants the medium of Murashige and Skoog (1962) has been the basal one in most research with diosgenine spp. Mascarenhans et al., (1976) used also White (1963) or Smith (1967) media in
combination with Murashige and Skoog ( (1962) basal medium. Some less common media have been used by some others (Chaturvedi and Chawdhury, 1980) when callus culture is considered.
The research for the best form and the best ratio of nitrogen compound in shoot and root organogenesis from tumor callus of D.deltoidea has been done by Singh (1978) and by Chaturvedi and Chawdhury (1980) for callus proliferation.
But the more frequent research in medium adjustment is the levels and the balance of auxins and cytokinins for shoot and root organogenesis (Lakshmi Sita et al., 1976, for instance).
The first goal of in vitro culture with food yams was an understanding of tuberisation through bulbil organogenesis in D. opposita and D. bulbifera. However, addition of growth substances in the mineral basal medium, which was sufficient for bulbil development, leads Uduebo (1971) to find the parts of the level and the balance of auxin (IAA) and cytokinin in callus, bulbil, shoot and root organogenesis.
In vitro culture of D. alata has succeeded also in the laboratories of Y. Demarly and R. Nozeran an at the university of Paris-Orsay (Arsene, 1979, Espiand , 1980).
In Dioscorea composita an in vitro clonal multiplication method via somatic embryogenesis has been developed by Viana and Mantell (1989) and complete plantlets can be obtained easily through nodal segment culture (Mantell et al., 1978).
2.10 Micropropagation of Yams
Plant tissue culture techniques have been successfully used for rapid clonal multiplication of high yielding genotypes (Mantell et al., 1978).
Clonal propagation from nodal and meristem cultures
A number of species such as D. alata, can be propagated in the greenhouse or field, albeit inefficiently, through nodal cuttings (Coursey, 1967b). One goal of investigations of Diooscorea and tissue culture research has been to utilize techniques of meristem culture (Hu and Wang, 1983) for large scale clonal propagation.
Chaturvedi and Srivastava (1976) showed that rooting of stem cuttings of D. deltoidea was difficult when compared to similar material from D. floribunda. Among auxins and cytokinins, kinetin proved to be effective in nodal cultures of D. floribunda (Lakshmi sita et al., 1976), (Mantell et al., 1980). Mantell et al., (1980) found BA to be preferable to kinetin in apical shoot tips of D. alata. As auxin level increases, there is a tendency for callus formation, e.g. D. alata (Ammirato, 1976) and D. bulbifera (Uduebo, 1971).
Chaturvedi (1975), in one of the first investigations reported that single- node cutting on medium with 0.5µ M NAA and adenine sulphate rooted, then formed some tuberous tissues with the development of one or two shoots.
Mantell et al., (1978) reported that one excised nodal segment of D.alata or D. rotundata would produce a complete plant in 3-5 weeks.
Meristem cultures also offer the prospect of germplasm storage by means of either minimal growth storage or cryopreservation (Chaturvedi et al., 1982). There has been some success with D. rotundata (Henshaw, 1982).
Nodal segments of D. alata regenerated into shoots on M.S. medium with 10-6 M NAA & 5 x 10-6 BAP. The regenerated shoots were micropropagated on M.S. medium without growth regulators. Microtubers are found to be produced in aging cultures of D. alata in M.S medium supplemted with 10-6 M NAA & BAP. These microtubers on transfer to fresh medium produced new shoots and roots (Nair, 1985).
White yam (D. rotundata) was cultured on M.S. medium supplemented with 10-6 BAP and NAA using nodal segments as explants (Nair, 1985). Axillary buds developed into shoots which on transfer to fresh medium developed into whole plants with well developed root system.
Shoot tips of D. rotundata responded well with regeneration on M.S media with NAA & BA with 1µM concentration. Meristem tips yielded only callus. (CTCRI, 1983).
Nodal stem segments of D. bulbifera were induced to form plantlets in vitro (Clare Forsyth & J Vanstaden, 1981) Rooted plantlets were obtained on M.S revised medium supplemented initially with 5 mg/l kinetin & subsequently with 5 mg/l IBA. By increasing the kinetin concentration from 5 mg/l- 10mg/l the number of shoots forming per node was increased when BAP (1mg/l) was added instead of kinetin, number of shoots produced was more.
D. bulbifera could be micropropagated through nodal segments on M.S medium supplemented with 0.5µM IAA, 200 µM kinetin, 500 mg/L casein hydrolysate and 20% activated charcoal. Diosgenin was maximum in regenerants grown as M.S with 5µM IAA, 20µM kinetin and 500 mg/L casein hydrolysate. (Narula, et al., 2003).
D. nipponica nodal cultures on M.S with 3% sucrose, 2mg/l BA and 1mg/l NAA gave highest frequency of shoot induction (Chen et al., 2007).
In vitro tuberisation/ in vitro bulbil formation
Nodal cuttings of white yam (D. rotundata Poir) produced microtubers on M.S revised medium supplemted wirh various concentrations of sucrose, 20 mg/l L- cysteine, 0.5 mg/L kinetin and 0.7% agar (Ng SYC 1988).
Bulbil development in cultured nodes of D. bulbifera produced in the absence of growth substances from the medium (Uduebo, 1971). When IAA was incorporated in to the medium at the concentration of 5 mg/l-10mg/l the cultured nodes produced larger bulbils than in its absences.
Nodal segments of both D.bulbifera and D. alata have produced tubers directly in culture (Ammirato, 1976, 1982). The Formation of small tubers on plants grown from nodal cultures and meristem cultures has been observed in D. floribunda ( Chaturvedi, 1975; Grewal et al., 1977).
Ammirato (1982) reported that both D. alata var, gemelos and D. bulbifera var. sativa plants, after growing for 4-5 months in continuous light, developed numerous aerial tubers. Lauzer et al., 1992 micropropagated two wild yams of West Africa D. abyssinica Hoch & D. mangemotiana Miege from nodal segments. In D. abyssinica media containing 20, 40, 60 and80 mg/l sucrose induce micro tuber production.
Microtubers were induced in M.S medium containing 4% sucrose, 2.5µM kinetin held under 8 hr photoperiod in D. bulbifera (Mantell and Hugo, 1989).Microtuber induction was observed in D. bulbiferaon M.S medium supplemented with NAA(0.1µM ),BA(0.5µM ),GA3(0.1µM ),sucrose 40mg/l and activated charcoal 1g/l after 5-6 months. Sreeja et al., 2005.
M.S media supplemented with various hormones induced tuber formation in D. alata(CTCRI,1989). Alizadeh etal., 1998, investigated the individual effects of sucrose, plant growth regulators and basal salt media formulation on microtuer indctions & development in shoot cultures of D. composita. Sucrose at 8% (w/v) was the single most significant medium tested. Of the four cytokinins tested, 6-benzyladenine at 1.25 and 2.5µM showed strong inhibitory effect on microtuber induction. The auxins,α-NAA &IBA at 5µM showed strong promotive effects on micro tuber induction and growth.
D.bulbifera could be micro propagated through bulbils. Best medium for this is MS+ 0.5µM IAA+ 200 µM kinetin+500 mg/L casein hydrolysate. T.S of bulbil also is used for direct plantlet differentiation and bulbil differentiation (Narula et al., 2003).
D.nipponica nodes were cultured on M.S media for micro tuber induction. Cytokinin (BA) in range of 0.5-2 mg/l showed strong enhancing effects or micro tuber induction in conjuction with NAA at 0.5-2 mg/l. Sucrose at 7% was the single most significant medium constituent for micro tuber growth. The heaviest micro tubers were formed on media containing 1 mg/l BA and 2 mg/l NAA especially with 7% sucrose (Chen et al., 2007).
Callus culture in Yams
Regeneration of plantlets from calluses are reported. Callus of D. floribunda and D. bulbifera initiated on M.S medium + 2-4 mg/l 2,4-D (and some times 0.2 mg/l BAP in addition) was found by Ammirato (1982) to produce somatic embryos when the cultures were aged or when the callus was transferred to a medium lacking 2,4-D but containing glutamine (500 mg/l) and zeatin (0.02mg/l) or ABA (0.03 mg/l).
Explants with various combinations of M.S medium with different levels of NAA, BA & GA3 on regeneration of shoot tips and nodal segments with dormant axillary buds were done to find out a suitable medium for D.esculenta. Excess callus development was observed in all combinations of medium with NAA & BA at 10.0 µM (CTCRI, 1983).
Regeneration of plantlets occurred from calluses obtained from zygotic embryos in D.composita Hemsl and D. cayenesis L. M.S basal medium supplemented with 18µM 2, 4-D favoured callus formation while plantlet regeneration occurred in media containing 0.1 µM zeatin and 3.3mM glutamine. Regeneration of plantlets from D.cayenensis calluses occurred only at low levels of 2, 4-D (2.25 µM) contained in the media (Mantell et al., 1980).
Segupta et al., (1984) obtained callus cultures from node and internode segments of D. floribunda. According to their work both M.S and modified Whites medium supported callusing and organogenesis along with either 2, 4-D or NAA in combin
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