Do you want to create an amazing science fair project on potatoes ? You are in the right place. Read the below given article to get a complete knowledge about:- 1. Origin of Potato 2. Production of Potato 3. Cytology 4. Potato Species 5. Botany 6. Breeding Uniqueness 7. Breeding Goals 8. Reproductive Biology 9. Hybridization 10. Breeding Methods 11. Biotechnology 12. Useful Donors and Others.
Contents:
- Science Fair Project on the Origin of Potato
- Science Fair Project on the Production of Potato
- Science Fair Project on the Cytology of Potato
- Science Fair Project on Potato Species
- Science Fair Project on the Botany of Potato
- Science Fair Project on the Breeding Uniqueness of Potato
- Science Fair Project on the Breeding Goals of Potato
- Science Fair Project on the Reproductive Biology of Potato
- Science Fair Project on the Hybridization of Potato
- Science Fair Project on the Breeding Methods of Potato
- Science Fair Project on the Biotechnology of Potato
- Science Fair Project on the Useful Donors of Potato
- Science Fair Project on Genetic Basis of Heterosis for Yield in the Autotetraploid Potato
- Science Fair Project on True Potato Seed (TPS) Technology
- Science Fair Project on Central Potato Research Institute (Cpri)-Shimla
- Science Fair Project on the Varieties of Potato
Science Fair Project # 1. Origin of Potato:
The potato is one of mankind’s ancient cultivated plants. Potato is an Andean tuber crop that was originally domesticated in South America and started its worldwide dissemination after Columbus’s voyages brought to Europe in the late 16th century some years after the discovery and conquest of Peru.
There are strong evidences that potato was widely distributed throughout the Andes, from Colombia to Peru and also in southern Chile. In Central America and Mexico, although wild species of the potato are native to these regions, the cultivated potato seems to have been a comparatively recent, post-Columbian, introduction.
Potato was commonly grown in Spain and Italy by the late 16th century. Bauhin sent potatoes to France by about 1600 and they were widely grown there by the mid-17th century. The Slavic nations received their potatoes from Germany.
The general adoption in eastern Europe was late 18th to early 19th century when the adaptation to short day length had been bred out of them. Even in the milder winter climate of England, potatoes did not become universally grown until the mid-18th to early 19th centuries.
However, the much milder climate of south-west Ireland allowed potatoes to be grown there in the early 17th century. By the mid-18th century, potato was taken to Norway and then to Sweden and Denmark from Scotland and to southern Denmark from Germany.
Potatoes in the North American colonies were first received from Bermuda in 1621 where they had been grown after an initial importation from England in 1613. No records exist of importations from South America until Goodrich obtained his varieties from Panama.
Potatoes are said to have been taken to India and to China by British missionaries in the late 17th century and were known in Japan and parts of Africa by about the same period. In New Zealand, they were introduced in 1769 and were adopted by the Maoris by 1840, who knew sweet potato cultivation.
With respect to tracing progenitor species from which the cultivated potatoes originated, the hypothesis of Vavilov (1951) is to be taken into account. According to this, the origin of a cultivated plant is to be found in its region of greatest diversity i.e. the centre of origin.
It is assumed that longer a crop had existed in an area, the greater would be its genetic diversity and must have evolved there from wild species among which the most primitive diploid species might have been the first to be domesticated.
Based on various considerations, Hawkes (1990) concluded that potato may have been domesticated in what is now the Lake Titicaca to Lake Poopo region of north Bolivia and it originated from the wild diploid species S. leptophyes some 10,000-7000 years ago, and the first domesticated species was Solarium stenotomum.
The evolution of S. stenotomum was only the beginning of potato evolution. In addition to first wild species S. leptophyes which gave rise to domesticated diploid species, S. stenotomum, 3 other wild species, namely, S. sparsipilum, S. acaule and S. megistacrolobum were instrumental in evolution of present day cultivated potatoes.
Some authorities believe that S. tuberosum is a straight tetraploid of S. stenotomum but there are stronger evidences in support of the allotetraploid origin of S. tuberosum by hybridization between S. stenotomum and S. sparsipilium. During the course of evolution diploid species S. megistacrolobum and the tetraploid species S. acaule contributed frost resistance.
A diploid cultivated species, S. phureja evolved from S. stenotomum by human selection exercised for rapid maturity and lack of tuber dormancy to develop varieties to be grown 2-3 times in a year in the lower, eastern frost free Andean valleys. The evolution of S. tuberosum ssp andigena is (1990).
Science Fair Project # 2. Production of Potato:
Potato (Solanum tuberosum, 2n = 4x = 48) is very widely grown on world scale, ranking fourth in food production after rice, wheat and maize (650, 625, 604 and 314 million tons, respectively). World acreage of potato is 18.2 million hectares with average productivity of 17.2 tons/ha.
Potato yields on average more food energy and protein per unit of land than cereals. The lysine content of potato complements cereal based diets that are deficient in this amino acid.
The major potato producing countries in the world are listed in Table 34.1:
India is the major potato growing country in the world and produces 34.39 million tons annually on 1.82 million hectares with the average productivity of 18.9 tons/ha as per production figures for 2008-09.
Commendable growth of potato crop in India has been on account of:
a. Intensive research and development efforts by Central Potato Research Institute, Shimla
b. Highest production of dry matter and protein per unit area and time by potato
c. High nutritional value of potato
d. Short duration crop fitting well in the intensive cropping system
e. Round the year cultivation in one part or other in India.
Major production trend in potato in India over the 9 years period is given in Table 34.2:
Major potato growing states in India are as follows:
a. Uttar Pradesh – 527300 ha
b. West Bengal – 400800 ha
c. Bihar – 310300 ha
d. Punjab – 81100 ha
e. Assam – 79700 ha
f. Karnataka – 71600 ha
g. Madhya Pradesh – 66200 ha
h. Gujarat – 57000 ha
Major potato producing belts in India are:
a. Himachal Pradesh (Shimla, Lahaul, Spiti, Mandi)
b. Punjab (Jalandhar, Hoshiarpur, Ludhiana, Patiala)
c. Haryana (Ambala, Kurukshetra, Hisar, Karnal)
d. Uttar Pradesh (Farrukhabad, Etowah, Manpuri, Barabanki, Allahabad, Badaun, Moradabad, Agra, Aligarh, Mathura, Faizabad)
e. Madhya Pradesh (Sidhi, Satana, Rewa, Sarguja, Rajgarh, Sagar, Tikamgarh)
f. Rajasthan (Bharatpur, Dholpur)
g. Gujarat (Khera, Dissa, Banashkestha, Jamnagar, Baroda, Mehsana)
h. Orissa (Cuttack, Dhenkanal, Puri, Sambhalpur and West Bengal)
i. Maharastra (Pune, Satara, Kochapur, Nasik)
j. Karnataka (Belgaum, Dharwad)
k. Andhra Pradesh (Medak, Chittur)
l. Karnataka (Hassan, Kolar)
m. Tamil Nadu (Dhindigulanna, Nilgiris)
Potato is grown in India in almost all the states under very diverse conditions. The states of UP, West Bengal and Bihar account for nearly 3/4th of the area and 4/5th of the total production under potato in India. The per capita availability of potato in India is 17.7 kg which is almost 1/3 of the world average.
The best U.S. farmers have recorded tuber yields in excess of 67 t/ha. The highest national average fresh tuber yield has been recorded in the Netherlands (44 t/ha), although the potential tuber yield of a potato ideo-type may exceed 100 t/ha in long-day temperate climates.
Conversely, about 10 t/ha is the national average tuber yield in Peru, which shows that higher yields are achieved outside the area of the origin of this crop.
The International year of potato was celebrated throughout 2008 aiming at raising global awareness of potato’s key role in agriculture, the economy and the world food security with focus on:
a. Breeding high yielding and disease resistant cultivars
b. Standardization of production practices and solution of problems related to dormancy and storage
c. Survey of diseases and pests and devising control measures
d. Production of nucleus and breeder seed
e. Organizing training programmes on different aspects of potato cultivation
Potato is included in the multilateral system established under FAO’s International Treaty on Plant Genetic Resources for Food and Agriculture. The treaty which entered into force in 2004, aims at conservation and sustainable use of crop plant diversity and the fair and equitable sharing of benefits derived from their use.
Science Fair Project # 3. Cytology of Potato:
Potato has basic chromosome number as 12 and right from diploid to hexaploid species are available. Majority (about 75%) of the species are diploid followed by tetraploids which are about 15%.
Triploid potato species are derived from spontaneous crosses between diploid and tetraploid species. Pentaploids are obtained from crosses of hexaploids with tetraploids. Triploids and pentaploids are highly sterile and are maintained by vegetative propagation.
There are 3 cultivated diploids, viz., S. stenotomum, S. phureja and S. ajanhuiri of which former two are sexually fertile and the latter one is less fertile and does not breed true. The two cultivated triploid species (S. chaucha and S. juzepczukii) are more or less sterile.
The cultivated tetraploid species (S. tuberosum ssp tuberosum and S. tuberosum ssp andigena) are usually fertile except in a number of highly bred clones outside South America. In pentaploid category, there is only one species i.e. S. curtilobum which is reasonably fertile in crosses with S. tuberosum, but not in self-mgs.
Regular meiosis has been observed in diploid, allotetraploid and allohexaploid species, but the tetraploid cultivated species, S. tuberosum behaves as autotetraploid cytologically, although there is considerable evidence that S. tuberosum evolved as hybrids between two somewhat closely related diploid species.
Nearly all the diploid species are self-incompatible while all the tetraploids and hexaploids are self-compatible. The F2 hybrids between diploids are obtained by F1 sib-matings as self-incompatibility does not allow selfing. Genetic breakdown is common in F2 generation.
Science Fair Project # 4. Potato Species:
Hawkes (1990) has mentioned the number of wild and cultivated potato species at various ploidy levels as given in Table 34.3.
The tuber bearing Series Tuberosa Rydb. contains all the cultivated potato species as well as the wild and weed species most closely related to them. This Series is characterized by imparipinnate or simple leaves, bifurcate peduncle, rotate to semi-stellate corolla and round berries.
This Series is found in the Andes of South America and adjacent coastal belt in temperate and sub-tropical latitudes. This contains 68 wild species and 8 cultivated species of which most common are tetraploids and are known as S. tuberosum L.
S. tuberosum L. is distinguished from other species of cultivated potato by the pedicel articulation placed in the middle third, short calyx lobes arranged regularly. The leaves are often slightly arched. Leaflets are always ovate to ovate-lanceolate, about twice as long as broad. Corolla lobes are about half as long as broad. Tubers are with well-marked dormancy period.
There are 2 subspecies under this species as given below:
Subspecies tuberosum:
It has originated from the coastal regions of South Central Chile. It is distinguishable from ssp. andigena by the less dissected leaves with wider leaflets, generally arched and set at a wider angle to stem. Pedicel is thickened above. Corolla is often white or pale coloured. Tubers are formed under long days or under short days in the tropics at lower altitudes only (500 – 2000 m).
This subspecies was derived from ssp. andigena probably on 2 separate occasions. First change occurred in Chile where ssp. andigena was carried by the Indian tribes migrating south wards from the Bolivian Andes.
Secondly, ssp. andigena was brought to Europe after the Spanish conquest where under similar climatic and day length conditions to those of Chile, the typical ssp. tuberosum was formed, again partly as a result of artificial selection. Initially, it was cultivated on the southern coast and islands of southern Chile but is now spread worldwide.
Subspecies andigena (Juz. et Buk.) Hawkes:
This ssp. is distinguishable by the narrower and more leaflets which are generally petiolate. The leaves are set at an acute angle to the stem and are generally more dissected. Pedicel is not thickened at apex. Tubers are formed at high altitudes only (over 2000 m) under short day conditions.
This is the ancestral ssp. of S. tuberosum formed probably from crosses of S. stenotomum X S. sparsipilum in the Andes of Peru and Bolivia, Andes of Venezuela, Colombia, Ecuador, Peru, Bolivia, N.W. Argentina and also sparingly in Guatemala and Mexico. For details on other species Hawkes (1978, 1990) may be referred.
Science Fair Project # 5. Botany of Potato:
Potato stolons are lateral shoots, usually from the most basal nodes below soil level. Typically they are di-ageotropic shoots with elongated internodes, hooked at the tip. They have spirally arranged scale leaves. Tubers develop from the sub-apical region of stolons. However, tuber formation includes 2 processes, viz., stolon formation and tuberization of the stolon tips.
Stolon formation usually begins at the lower nodes and progresses acropetally. The first tubers, in turn, usually develop from the lower stolons and tend to become dominant over those formed later. Tubers are important as 75-85% of the total dry matter produced by the plant is stored in them. Potato tuber is a modified stem with a shortened axis and rather poorly developed leaves.
The ‘eye’ of the potato tuber is a leaf scar with a subtended lateral bud having undeveloped internodes. Tuber formation occurs earlier at low temperatures and gets delayed at high temperatures. Tuber yield is good if plants are grown in short days with low night temperatures.
No tubers are formed in short days with high night temperature. In the cultivated potatoes, plants maintained under long days make active vegetative growth and ultimately form tubers, some 3-5 weeks later than plants kept under short days.
The inflorescence of potato is cymose. The vegetative shoot is a sympodium, each portion terminates in an inflorescence, vegetative growth being continued by the bud in the axil of the last true foliage leaf. This shoot appears to be laterally displaced. The flowers are actinomorphic and hypogynous. Calyx has 5 lobes. Corolla tube consists of 5 petals.
The 5 stamens alternate with the petals and are borne on corolla tube. The anthers are fused and enclose the pistil. At maturity the stamens have short, stout filaments and long anthers.
Pollen is shed through pores at the tips of the anthers. Two carpels fuse to form a syncarpous, bilocular, superior ovary with long style and a stigma with 2 lobes. The mature fruit is a green berry with axile placentation, which often fails to develop in a cultivated potato.
Flowers in the cultivated potato open mostly in early morning, although a few may continue to open throughout the day. Self-pollination in nature is the rule. Cross-pollination is most often accomplished by bumblebees, which are the main carriers of pollen. Wind-pollination plays a minor role in nature.
Germination of the pollen is completed after 30 minutes, and the ovary is fertilized within 12 hours. Obstacles to seed production in the potato include:
a. Failure to flower
b. Dropping of buds and flowers either before or after fertilization
c. Low pollen production and failure to produce viable pollen
d. Male sterility, and
e. Self-incompatibility
Science Fair Project # 6. Breeding Uniqueness of Potato:
a. Propagated asexually
b. Transmission of diseases via tubers
c. Easy maintenance and multiplication of elite material in original genetic state through vegetative propagation
d. Complex tetrasomic inheritance due to autotetraploidy
e. Diverse source of germplasm including wild relatives for resistance to abiotic and biotic stresses
Science Fair Project # 7. Breeding Goals of Potato:
1. High tuber yield
2. Earliness
3. Photoperiod insensitivity
4. Responsiveness to fertilizer
5. Better keeping quality (resistance/tolerance against shrinkage, rottage, accumulation of sugars, specially reducing sugars and reasonable dormancy)
6. Better quality tubers:
(i) Round, medium sized with shallow eyes and free from greening for general consumption
(ii) High vitamin C and protein content
(iii) High specific gravity (dry matter content) suitable for French fries, chips and dehydrated products
(iv) Low sugar content for chips and French fries to avoid browning
7. Resistance to:
(i) Late blight (Phytophthora infestans)
(ii) Early blight (Alternaria solani)
(iii) Charcoal rot (Macrophomina phaseolina)
(iv) Wart (Synchytrium endobioticum)
(v) Common scab (Streptomyces scabies)
(vi) Bacterial wilt (Pseudomonas solanacearum)
(vii) Soft rot (Erwinia carotovora)
(viii) Viral diseases (potato virus X, potato virus Y)
(ix) Nematodes (cyst nematode – Globodera rostochiensis, G. pallida), root knot nematode (Meloidogyne incognita)
8. Resistance/tolerance to aphids (Myzus persicae), potato tuber-worm (Phthorimaea operculella)
9. Resistance/tolerance to heat, drought, frost, soil salinity
Science Fair Project # 8. Reproductive Biology of Potato:
The details under this section are primarily based on Howard (1978) and Kang and Birhman (1993).
Flowering:
Potato flowers under long days (around 16 hrs.) moderate temperature and high humidity. Such conditions are available in the hills of northern India where potato is grown under long summer days, and abundant rainfall and humidity.
Although, most of the potato genotypes flower under such conditions, however, some genotypes do not flower even under these conditions. Various methods, such as planting on bricks, removal of tubers and spray of gibberellic acid have been suggested for induction of flowering in non-flowering genotypes.
In ‘growing on a brick’ sprouted tubers are planted on a brick above soil level and covered with sand. When roots have grown through the sand into the soil, the sand is washed away. This allows new tubers to be removed when very small. Removal of young tubers results in no sinks for assimilates and hence the flower buds receive more nourishment.
Sterility:
Complete or partial male sterility resulting from complete absence of poor quality pollen is frequent in cultivated tuberosum and andigena potatoes. This reduces the choice of pollen parents in potato breeding. South American wild and cultivated Solanum species have high degree of pollen fertility.
Incompatibility:
Self and cross incompatibility operating in an oppositional factor system and conditioned by a series of multiple alleles is widely prevalent in diploid Solanum species. Incompatibility creates problem in tapping diploid Solanum species for potato improvement, which otherwise possess many valuable traits like resistance/tolerance to biotic and abiotic stresses.
But absence of self/cross incompatibility in some genotypes of a species and its non-expression under certain environmental conditions, however, provides an opportunity to secure seeds where normally no such seed production is possible.
Cross-ability:
Tuberosum and andigena potatoes are freely crossable. But, potato germplasm possesses a vast reservoir of related semi-cultivated and wild species. To formulate a breeding programme, crossing behaviour of these species among themselves and with tuberosum and andigena should be studied.
Berry Setting and Maturity:
Berry drop is a common problem in potato hybridization. Spraying of 2, 4-D solution (250 ppm) or other auxin analogues 2-3 days after pollination may prevent the abscission of flowers and allow ovaries to develop into berries with seeds.
Science Fair Project # 9. Hybridization of Potato:
Although self-fertilization is possible in some varieties, the breeders generally prefer to cross two parents with a view to combine their desirable characters. Hybridization technique in potato has been standardized and described by Pushkarnath (1960). According to Poehlman and Sleper (1995), flower buds that are mature are selected for emasculation just prior to crossing.
It is particularly important to emasculate just prior to crossing if pollinations are done in the field as the wind can break off the stigmas before pollination occurs if they are emasculated too far ahead of pollination. Mature buds are plump, with the petals ready to separate. The remaining buds and opened flowers in the bunch are removed to facilitate emasculation of the selected buds and to prevent contamination of the emasculated flowers by the open flowers.
There is a limit to the number of flowers from an inflorescence that will set fruit/seed, so removing the extra flowers increases the chances that the pollination will be successful.
The petals of the selected flowers are gently pushed apart along the sutures and the five stamens removed with fine-pointed forceps without breaking the style. The emasculated flowers are then bagged. Inserting a branch with one or two leaves into the bag helps in maintaining a humid climate inside the bag. In fully self-sterile parents, emasculation is unnecessary.
Pollination can be done at any time of the day so long as the temperature is not too high. Open flowers are collected from the plant to be used as a male. The flowers are laid out to dry overnight.
The following morning the pollen is collected from them by shaking into gelatin capsules such as those used in the pharmaceutical industry (other small tubes can also be used). For large quantities of flowers, the pollen is shaken out by placing the flowers in the top section of a sieve, and the sieve is then shaken at high speed.
The pollen falls through and is collected in the bottom chamber of the sieve and transferred to the smaller capsules or tubes for storage. Pollen can be stored desiccated in the refrigerator for 1 to 2 weeks and in the freezer for 6 months to a year.
To make the pollination, the stigma is dipped in the pollen in the capsule or tube, and then the pollination tag is attached and the bag is placed over the flower and left on until the fruit is harvested. Setting of seed may be observed in about 7 to 10 days.
Average seed set per berry varies with the cultivar, but levels of 50 to 200 seeds per fruit may be obtained. The seeds are extracted from ripened berries by macerating in water and washing.
Science Fair Project # 10. Breeding Methods of Potato:
Potatoes are vegetatively propagated and therefore their breeding is considered to be easier than that of crops with sexual reproduction in one important aspect. That is that any selection will keep true to type, except for rare mutations, as long as vegetative reproduction is allowed.
New varieties may be selected as plants from parental crosses in the F1generation. F1generation itself provides enough variability due to heterozygosity of parental cultivars and involvement of autotetraploidy. However, selection of promising parental cultivars for production of F1 progeny in which desired characters are to be combined, is difficult task. Four main types of parents are available to the potato breeder.
They are:
(i) Tuberosum varieties
(ii) Andigena varieties
(iii) Varieties of cultivated diploid species
(iv) Wild species
It is now generally agreed that for most parts of the world a Tuberosum variety and able to produce a high yield under long-day conditions, should always be one parent of a cross and that where a desired character cannot be found in the Tuberosum varieties, the next source should be an Andigena variety, cultivated diploid potato and wild species in this order.
Less favourable characters are difficult to be completely avoided. In order to avoid the recurrence of ‘wild’ characters in the progeny, potato breeders usually do not use unimproved materials particularly the wild species in the crossing programme.
It is safer to use commercial varieties and advanced breeding lines as parental cultivars. While planning a crossing programme, it should be kept in mind that many varieties are male sterile and hence selection of male parent is troublesome. In India crosses are normally made in open fields at Kufri in Shimla hills although indoor hybridization is also possible.
Raising and Selecting Seedlings:
It is usual now in most countries for potato seedlings to be grown in pots in aphid-proof glasshouses. Seeds are sown in pans and seedlings pricked out into the pots when large enough. Optimum temperature for true potato seed germination is 18 – 20°C. Germination is adversely affected at temperature beyond 25°C.
For potato breeding for plains in India, F1seedlings are raised at 3 regional stations of Central Potato Research Institute (CPRI) located in 3 different agroclimatic zones, namely, Jalandhar (western Indo-Gangetic plains), Modipuram (central Indo-Gangetic plains) and Patna (eastern Indo-Gangetic plains). Common size pots are 10 cm in diameter.
Smaller pots allow more seedlings to be grown but the tubers are rather small. Large pots will facilitate larger tubers to be produced, but number of seedlings/unit area is reduced. Because of the heterozygous nature of potato and the requirement of combining several desirable traits into one variety, it is usual in potato breeding to grow many thousand seedlings each year.
This large number of seedlings necessitates retaining only one tuber of each selected seedling. Selection in seedling generation is usually not too stringent because performance in the seedling year may not be closely related to performance in later clonal years due to the fact that plants are grown from seed and not tubers and the plants are grown in small pots. However, too ‘wild’ looking seedlings (with very long stolons and very late maturity) or too weak ones may be discarded.
Selection in the First Clonal Year:
Normally tubers of selected seedlings are planted in field. Selecting the best clones in the first clonal year is difficult due to the fact that there is only one plant/clone and big errors might creep in when yield is determined on single plant basis.
However, selection in early years is effective for characters with high heritability. Therefore, it may be possible to select the first clonal year plants efficiently for characters such as tuber shape and depth of eye.
Selection in Later Clonal Generations:
As the number of selections retained decreases, the number of plants/selection is increased in successive years. Once the clones contain a sufficient number of plants, selection for yield, resistance to diseases and pests and for quality are carried out. A representative scheme used at Plant Breeding Institute, Cambridge as outlined by Howard (1978) is given in Table 34.4.
Use of Dihaploids in Potato Breeding /Analytical Breeding Scheme:
The traditional breeding procedure in potato is to intercross superior autotetraploid parents and select F1 individuals. Such selection is laborious and the chances of finding a superior new recombinant are remote as on meiosis the optimally balanced, highly heterozygous genotype of each parent divides into numerous, and diverse male and female gametes.
In a cross combination, these are fertilized by a completely random process. Efforts to induce desirable changes in useful varieties by mutagenic treatment, leaving the overall genotype largely intact, have not been encouraging. Further narrowness of the genetic base of the commercial tuberosum potatoes is another reason for relatively slow progress in conventional potato breeding.
A potato breeding scheme based on di-haploids has been advocated in overcoming some of these constraints in potato breeding.
The essential steps in such a scheme are:
(i) Raising plants from gametes (= di-haploids) of tuberosum and andigena potatoes
(ii) Vegetative propagation of the di-haploids to enable a reliable evaluation of these genotypes for various kinds of resistance, yielding ability and quality characters
(iii) Hybridization of di-haploids with different semi-cultivated and wild diploid species to incorporate the desirable traits such as resistance to biotic stresses, genetic diversity and quality traits
(iv) Sexual tetraploidization using 2n gametes formation in di-haploid species hybrid through unilateral (Ax – 2x or 2x – Ax) and bilateral (2x – 2x) matings
There are three essential components to this breeding method:
(a) Wild and cultivated relatives provide genetic diversity,
(b) Di-haploids of tuberosum and andigena effectively capture this genetic diversity and put it into usable form, and
(c) 2n gametes effectively and efficiently transmit this diversity to cultivated Ax potatoes.
These Ax genotypes are allotetraploids and are called meiotic tetraploids, to distinguish these from autotetraploids, produced from di-haploids through mitotic manipulations. The practical utility of this nonconventional method of potato breeding is yet to be realized on meaningful scale.
Induced Mutations:
Potato was considered to be an ideal crop plant for improvement through induced mutations, because desirable mutants once identified could be easily maintained. But, the efforts made in this direction have not met with much success, probably, due to its tetraploid nature and high intrasomic competition, resulting in low frequency and narrow spectrum of mutations (1/200000- 500000).
Induced mutations isolated, by using various types of chemical and physical mutagens, were either not useful, or when useful, either reverted or were agronomically inferior and were, therefore, discarded. Most somatic mutations are deleterious and are rogued out. Very rarely they may give rise to new varieties.
Science Fair Project # 11. Biotechnology of Potato:
Potato is an ideal material for application of biotechnological methods, such as anther culture, somatic hybridization, genetic transformation, in vitro selection, somaclonal variation and production of transgenic. The potato has many pests and pathogens that can reduce yields and overall plant vigor.
The Colorado potato beetle (Leptinotarsa decemlineata) is a highly destructive pest of potato in north central and eastern North America, Europe, and Asia. Defoliation by adults and larvae can reduce yields and even result in total tuber loss. Despite breeding efforts, no potato cultivars with demonstrated resistance to Colorado potato beetle have been released commercially.
The bacterium, Bacillus thuringiensis (Bt) ssp. tenebrionis Berliner, produces a Cry 3A protein that has toxic effects on Coleoptera, including Colorado potato beetle. The choice of a Bt gene for engineering host plant resistance has multiple advantages.
Bt proteins have very specific modes of action, such that a protein with specific toxicity towards Coleoptera would not be toxic to other orders of insects; and Bt crystal proteins have not shown any toxicity towards humans, other mammals, or birds.
Synthetic Bt-cry3A genes have been constructed specifically to optimize expression in plants. The ability to transform and express Bt in plants provides a high-dose strategy of deploying Bt toxins for specific insect control.
Coombs (2002) developed transgenic potatoes containing Bt-cry3A for three potato lines with differing levels of resistance to Colorado potato beetle [‘Yukon Gold’ (susceptible control), USDA 8380-1 (leptine glycoalkaloids), and NYL 235-4 (glandular trichomes)].
Polymerase chain reaction, and Southern and Northern blot analyses confirmed integration and transcription of the cry3A gene in the transgenic lines. Detached-leaf bioassays of the cry3A engineered transgenic lines demonstrated that resistance effectively controlled feeding by first instar Colorado potato beetles.
The susceptible ‘Yukon Gold’ control suffered 32.3% defoliation, the non-transformed high foliar leptine line (USDA 8380-1) had 3.0% defoliation, and the non-transformed glandular trichome line (NYL 235-4) had 32.9% defoliation.
Mean percentage defoliation for all transgenic lines ranged between 0.1% and 1.9%. Mean mortality ranged from 0.0% to 98.9% among the Bt-cry3A transgenic lines, compared to 20% for the susceptible ‘Yukon Gold’ control, 32.2% for USDA 8380-1, and 16.4% for NYL 235-4.
Results indicate that genetic engineering and the availability of natural resistance mechanisms of potato provide the ability to readily combine host plant resistance factors with different mechanisms in potato.
After more than 10 years of research, scientists have developed experimental improved seed potatoes that are protected from serious pests, including insects and disease. The first commercial products resulting from this effort were New Leaf potatoes derived from both ‘Russet Burbank’ and ‘Atlantic’ parents.
The New Leaf product was developed in 1995 and contains the cry3A gene from Bacillus thuringiensis (variety tenebrionis) (B.t.t.), for the production of the Bt protein. Potatoes expressing this gene are completely protected from the Colorado potato beetle (CPB) and need no applied chemical protection for this insect pest.
The U.S. Food and Drug Administration, U.S. Dept. of Agriculture and U.S. Environmental Protection Agency have all determined that these potatoes are the same in safety and nutritional composition as any other ‘Russet Burbank’ and ‘Atlantic’ potatoes. These potatoes have also been approved by Health Canada, Agri-Food Canada, and Agriculture Canada and by Japan and Mexico for food use.
Trait stability, as reflected in year-to-year consistency in gene expression level and protein efficacy, is critical to the successful commercial application of genetically modified crops. Commercial grower across North America have experienced outstanding performance while growing New Leaf potatoes 5 years in a row.
This level of performance is the result of stable, non-significant differences in expression of the cry3A gene. The stable performance, also, is a result of an effective insect resistance management program based on maintaining CPB refuges near New Leaf fields, reducing CPB populations, and monitoring for CPB surviving exposure to New Leaf potatoes.
A similar degree of trait stability, but only over a 7 year study period, has been recorded for PVY resistance and PLRV resistance. This transgenic trait stability has been noted in 12 different transgenic potato products represented by 6 different potato cultivars.
Thus, New Leaf, New Leaf Y, and New Leaf Plus demonstrate the genetic stability required of viable commercial products. In 1998 New Leaf Y conferring resistance to both CPB and potato virus Y, and New Leaf Plus, conferring resistance to CPB and potato leaf roll virus were reported.
Transgenic potatoes containing high protein genes from amaranth have been developed in India by Chakraborty (2000). However, these are not yet commercialized. Potato was one of the first crop plants in which plants were successfully regenerated. Using recombinant gene technology, genes of interest are substitute to oncogenes and some new traits were introduced in potato cultivars.
For almost all transgenic potato plants developed today kanamycin resistance has been used as the selectable marker gene for the selection of transformed events. Different transformation protocols have been developed using other selectable markers or potato organs. The following scheme presents briefly the steps for agrobacterium mediated potato transformation on leaves and internodes (CIP protocol).
Somaclonal variation frequency, integration and expression of transgenes are very different among transgenic lines. Integration of the transgene may occur in a complete, truncated or arranged manner, as a single or various copies in the genome.
The level of expression of a transgene often varies among transgenic lines because of the effect of the chromosomal location “position effect” or either co-suppression or gene silencing effects. By generating a large number of transformed events and by further selection these effects can be circumvented in potato.
However, variation in transgene expression, down to silencing have a minor importance in potato crop. The agro infection system favors low copy number of transgene and clonal propagation will maintain the hemizygosity of transgenic lines with appropriate levels of expression.
Genetic engineering offers huge opportunities for the introduction of new genes into potato cultivars. Potato has been at the forefront of the development of genetic engineering in crop plants, with many public research institutes and private companies targeting potato improvement via the transformation of existing cultivars with specific genes.
This is a direct consequence of the importance of the potato crop throughout the world, the relative ease with which the crop can be transformed and genetic limitations associated with traditional potato breeding.
One of the major difficulties associated with traditional breeding relates to the tetrasomic nature of inheritance, virtually all the potato cultivars are autotetraploids (2n = 4x = 48). High heterozygosity and severe inbreeding depression in parental clones require exceptionally large populations of potato seedlings to be screened in order to recover individuals for evaluation as potential clones.
Thus, initial selection for desirable characters can often be inefficient and time consuming. In potato it may take up to 20 years to develop a new variety with conventional breeding, compared to just 3 years with genetic engineering
The second limitation of this system resides in the fact that with cross-pollination many unwanted traits are combined in the progeny along with the desired traits. The process of getting rid of all unwanted traits causes plant breeding to usually require several different backcrosses.
The development of resistance to pest, disease, frost and some quality traits such as content of low reducing sugar, starch and protein, has been greatly assisted by the transfer of genes from related Solanum species.
However, wild species also carry many other undesirable characters such as poor tuber shape, deep eyes on tubers and low yield. In many respects, the precise manner in which genetic engineering can control the nature and expression of the DNA to be transferred offers greater confidence for producing the desired outcome compared to traditional breeding.
Thirdly, cross-pollination is only possible with the same or very closely related species. There are several examples of Solanum species used for the introgression of gene into potato via traditional breeding methods.
However, in many occasions; the desired traits are not available within the species or any of the very closely related species Often, when the trait is available it is highly polygenic and has a low heritability making progress extremely slow. Through genetic engineering, researchers can combine genes from a wide variety of organisms reaching beyond the borders of the species or closely related species.
Although potato transformation can be accomplished by direct uptake of DNA into protoplast, Agrobacterium mediated transformation using binary vectors is the preferred method and is performed routinely in many laboratories.
Gene transfer into potato via agro infection is efficient, easy and inexpensive As the level of transgene expression differs among transgenic lines because of the chromosomal location (positional effects), numerous transgenic lines have to be produced and screened for desirable expression characteristics.
Concluding, the following table shows limitations and advantages of gene technology in potato as a tool for plant improvement in comparison with conventional breeding:
Examples of Solanum species used for gene introgression in potato are given in Table 34.5.
Science Fair Project # 12. Useful Donors of Potato:
Useful traits found in typical cultivated autotetraploid potatoes have been compiled by Kang and Birhman (1993) and Khushu and Birhman (1993). Based on these information’s, the useful donors are listed in Table 34.6.
Sources of Resistance in Wild and Other Species:
S. demissum shows high percentage of vertical and horizontal resistance to late blight. It is worth noting that out of 235 species of wild and cultivated potatoes, only 13 have actually contributed genes to potato cultivars.
The six wild species incorporated into European cultivars are as follows:
(i) S. demissum (late blight, potato leaf roll virus)
(ii) S. acaule (PVX, PLRV, potato spindle tuber viroid)
(iii) S. chacoense (PVA, PVY, late blight, tuber moth)
(iv) S. spegazzinii (Fusarium, wart, cyst nematode)
(v) S. stoloniferum (PVA, PVY)
(vi) S. vernei (high starch)
The important species carrying resistance to major potato diseases, pest and abiotic stresses based on Hawkes (1990) and Khushu and Birhman (1993) are listed in Table 34.7.
Science Fair Project # 13. Genetic Basis of Heterosis for Yield in the Autotetraploid Potato:
The cultivated autotetraploid potato is considered to be an out- breeder species which suffers from inbreeding depression and naturally expresses heterosis upon crossing of suitable parents. The two main hypotheses, i.e. dominance of favourable alleles and over-dominance, although conflicting, are not mutually exclusive.
Extensive reviews have been published on this subject but basically only diploid organisms have been considered. The genetic basis of heterosis in autotetraploids has received much less attention. Mendoza and Haynes (1974) have proposed a model of over-dominant gene action to explain heterosis for yield in the autotetraploid potato.
Loci with multiple alleles and a maximum heterotic value for quadrigenic genotypic structures have been postulated. Various experimental results have been analyzed on the basis of such a model in contrast with a dominance situation. The analysis suggests a close positive correlation between heterozygosity and yield.
The implication of the proposed over dominance model to potato breeding would be that substantial genetic advance in yield should be made upon increasing the genetic diversity of the parental clones. However, the alien sources of germplasm should undergo some previous selection for adaptation.
A proper balance between heterozygosity and adaptation, mainly to photo-period, should maximise the heterosis for yield in potato. Poehlman and Sleper (1995) have discussed this issue explicitly.
According to them the tetraploid nature of potato can be exploited by the breeder to improve desirable characteristics. It is well known that asexually propagated species such as potato have evolved talking advantage of non-additive or epistatic gene action.
Therefore, the potato breeder must be knowledgeable on the use of breeding procedures that can accommodate non-additive gene action. Because of the potato’s autotetraploid nature, intra-locus interactions (heterozygosity) and inter-locus interactions (epistasis) are important when selecting breeding procedures to improve certain traits.
It is assumed that increased heterozygosity leads to increased heterosis. Heterosis in potato occurs when the progeny outperforms the best parent or the parents’ mean. The level of heterozygosity is influenced by how different the four alleles are within a locus. The more diverse the alleles are within a locus, the higher the heterozygosity and the greater the number of increased inter-locus or epistatic interactions.
To see how increased heterozygosity can lead to more epistatic interactions, it is necessary to identity the allelic conditions possible in an autotetraploid. Five tetrasomic conditions are possible at an individual locus in an autotetraploid.
These are:
i. a1a1a1a1, a mono-allelic locus where all alleles are identical.
ii. a1a1a1a2, an unbalanced di-allelic locus where two different alleles are present in unequal frequency.
iii. a1a1a2a2, a balanced di-allelic locus where two different alleles occur with equal frequency,
iv. a1a1a2a3 a tri-allelic locus where three different alleles are present.
v. a1a2a3a4 tetra-allelic locus where four different alleles are present.
It is hypothesized that the tetra-allelic condition provides the maximum heterosis because more inter-locus interactions are possible for this tetrasomic condition than for the other configurations (Table 34.8).
For example, in the tetra-allelic condition, following interactions are possible:
i a1a2, a1a3, a1a4, a2a3, a2a4 and a3a4 = 6 first order interactions
ii a1 a2 a3, a1 a2 a4 , a1 a3 a4 and a2 a3a4 = 4 second order interactions
iii. a1 a2 a3 a4 = 1 third order interaction
Thus, is a total of 11 different interactions are possible for the tetra allelic condition. This is in contrast to the mono-allelic condition, which has no interactions. The highest level of heterosis will occur as the frequency of tetra-allelic loci increases.
The greatest number of inter-locus or epistatic interactions will also occur as the frequency of tetra-allelic loci increases. In breeding for improved tuber yield in potato, intra-and inter-locus interactions have been shown to be important. Procedures that maximize the frequency of teraallelic loci should be considered in breeding potato for increased yields.
Science Fair Project # 14. True Potato Seed (TPS) Technology:
To increase potato yield per unit area, new technologies will always be sought after. One of the constraints for potato production in India and other developing countries is the inadequate supply of healthy seed tubers at an affordable cost.
This problem could be overcome to some extent by using true potato seed (TPS). This concept was first realized to raise commercial crop in India by Dr. S. Ramanujam, the first Director of the Central Potato Research Institute in early fifties then at Patna.
The advantages conceived by him were:
(i) Requirement of TPS in small quantity and diversion of tubers meant for seed towards human consumption
(ii) Freedom of TPS crop from viral diseases common in seed tuber crops
(iii) Elimination of storage losses in seed tubers
Earlier investigations in this direction in India were initiated with open-pollinated seeds of an indigenous variety Phulwa (andigena group). However, this could not be accepted due to problems of crop heterogeneity and maturity. To overcome these problems, the approach of producing uniform homogeneous inbreds and then the F1 hybrids was pursued.
This also failed to give the desired results due to:
(i) Pollen sterility
(ii) Substantial inbreeding depression in the inbreds
Undeterred by these initial setbacks, a regular TPS research programme was started at CPRI, Shimla in 1976 and the researches during 1977-1984 yielded superior TPS populations. Out of about 200 lines screened, EX/A-680-16 has been found to be a good combiner.
During the latter half of eighties, large number of agronomical trials on TPS both as seedling transplants and first generation seedling tubers (F1C1) were conducted at Modipuram, Patna and Ooty stations of the CPRI.
Later on research work on TPS has been jointly conducted by the Central Potato Research Institute, the International Potato Centre and the All India Coordinated Potato Improvement Project resulting into development and identification of high yielding TPS populations, viz., TPS C 3 and TPS C 17 from Patna and HPS 1/13, HPS 2/67 and HPS 7/67 from Modipuram. It has been largely concluded that the transplanted seedlings in the field and use of seedling tubers as seed are successful approaches to commercial potato production.
This technology is likely to get momentum in primary target areas, viz., Karnataka, Maharashtra, Madhya Pradesh, Orissa and north-eastern hill regions where good quality tubers are either not available or cannot be produced.
The technology could further be expanded in the areas where good quality seed tubers can be produced but the shortage of breeder/foundation/certified seed necessitates continuation of degenerated seed.
For this technology to be of wider application, research efforts are needed on the following lines:
(i) Development of superior parental lines which would bloom profusely and set berries/ seeds both in the hills and plains
(ii) The male parent should be highly fertile and should produce pollen in abundance
(iii) The female parent should be male sterile
(v) The promising TPS populations should produce seedlings with vigour and capacity to bear transplanting shocks and
(vi) TPS should give rise to high yielding and relatively homogeneous population.
Production of Hybrid Tps:
The details on this have been provided by Grewal (1990) and Gaur (1991).
The technique in brief is as follows:
The parental lines are planted as summer crop in the northern hills. In plains, this is possible by providing 4-5 hrs. of extra light at the end of day during December-January by 150 W sodium vapour lamp (one/100 m2). FYM @ 20 t/ha is applied and mixed in the soil.
Male and female lines are planted in two separate but adjacent plots. The ratio of male: female is about 1: 4. Male block is planted about a week before planting female block at a spacing of 60 x 20 cm. Female block consists of beds of 3 rows (40 – 50 * 15 cm) having 80 cm walking space between 2 adjacent beds. The tuber size is about 30 g. After germination, only single stem/plant is maintained in the female block.
Before preparing a plant for pollination, old flowers and berries are removed. Bunches of flowers in these female plants are also trimmed to retain 5-6 large sized buds/bunch. All these are done 1 day prior to pollination. Freshly opened flowers/large sized buds which would open next day are collected from male parent in the evening preceding the day of pollination.
They are spread on a sheet of paper on a table. Next morning pollen are extracted in a small dish by shaking anthers using motorized mini mixer, electric buzzer or manually using a pair of forceps. Female flowers are pollinated applying pollen to the stigma using a brush in the morning followed by re-pollination next day.
Well-developed berries 45-50 days after pollination are collected and allowed to ripe at room temperature for 2-3 weeks. Ripe berries are crushed (in screw type juice extractor/low speed food blender) and seed and pulp are treated with 10% HCl with continuous stirring for 20 minutes.
Seeds are washed thoroughly with water and dried in shade in the plains or in mild sun in the hills. Dried seeds are kept in moisture proof containers and stored at low temperatures.
Crop Raising Using Tps:
Two methods are in use:
(i) Using seedlings as planting material
(ii) Using seedling tubers as planting material
In the first method 100-120 g TPS sown in a nursery of 50-60 m2 are required to raise seedlings for transplanting 1 hectare. Nursery bed width is kept as 1 m and length as per convenience.
The nursery bed is bordered using bricks up to a height of 10 cm. A substrate is filled in the bed to a thickness of 7-8 cm. The substrate consists of sterilized soil and well rotten FYM or compost or biogas slurry in ratio of 1: 1 plus 4-5 g N, 6-8 g P205 and 10 g K20/m2.
The top 2-3 cm of nursery bed is covered with sieved FYM. The TPS is soaked in 2000 ppm GA3 solution (200 mg GA3 dissolved in few drops of alcohol and made up to 100 ml by water) for 48 hrs. to break dormancy in freshly extracted seed. Seeds after drying in shade are sown in the early rabi season in the nursery bed in furrows (0.5 cm deep) 10 cm apart or seeds could be broadcast @ 2-3 g/m2 followed by covering with about 0.5 cm thick layer of sieved FYM. Soil is kept moist with light spray irrigation. After germination, seedlings are sprayed every 2-3 days with 0.1% urea till they are ready (4-5 leaf stage) for transplanting.
For transplanting the seedlings, the field is prepared in usual way and 20 cm high ridges, 40-45 cm apart are made in East-West direction. Before transplanting, the furrows are irrigated up to 8-10 cm depth and seedlings (one seedling/hill)are transplanted after irrigation at 10 cm intra-row distance at the water mark on the northern side of the ridge.
Second irrigation is given immediately after transplanting. Irrigation and transplanting are successive operations and are done row by row.
Later on irrigations are given as per requirement. After 30-35 days, earthing along with required nitrogen is done in such a way that the seedlings come to lie in the centre of the ridge. After harvesting, tubers are graded and 10-40 g size tubers are used as seed next year and those with more than 40 g weight are disposed off for table purpose.
In the second method of raising commercial crop from seedling tubers, seedling tubers are produced either in primary beds (10 x 10 cm) or the seedlings are raised in primary beds and subsequently transplanted in secondary (production) nursery beds for tuberization.
About 40 g TPS and 300 m2 nursery bed are required to produce enough seedling tubers for planting one hectare area next year. The requirement of seedling tubers to plant 1 ha area is as given in Table 34.9.
Isolation Distance:
1. Seed tubers-5 m for breeder/foundation certified seed
2. TPS-About 50 m
Science Fair Project # 15. Central Potato Research Institute (Cpri)-Shimla:
Potato research in India formally began on April 1,1935 with the opening of a Potato Breeding Station at Shimla and two seed production farms at Bhowali (Kumaon hills, UP) and Kufri (Shimla hills, HP) as a part of the Indian (then Imperial) Agricultural Research Institute. Delhi.
In 1945, a scheme for establishment of a Central Potato Research Institute (CPRI) was drawn up under the guidance of the then Agricultural Advisor to the Govt. of India, Sir Herbert Steward. Dr S. Ramanujam. who was then working as an Economist Botanist at 1ARI was appointed as an Officer on Special Duty for implementing the scheme in 1946.
It took more than three years for the scheme to attain concrete shape in the form of the Central Potato Research Institute at Patna in August, 1949. The Government of Bihar provided a 10 ha piece of land with an old single storey barrack-type building.
Three small units under the IARI looking after potato, namely Potato Breeding Station at Shimla, Seed Certification Station at Kufri and Potato Multiplication Station at Bhowali were merged with the newly-created Central Potato Research Institute and Dr. S. Ramanujam took over as the first Director.
The headquarters was shifted from Patna to Shimla in 1956 because the hills were the only source of disease free seed and the ideal location for hybridisation. All potato genotypes flower under long days of the hills and, therefore, more genotypes could be used for breeding new varieties.
Potato is not native to India. It was introduced in the country from Europe in the beginning of early 17th century. During 1824 to 1939 systematic attempts were made to introduce new potato varieties in the country, mainly from Europe.
These, however, could create very little impression as most of these varieties either failed to yield well under Indian conditions or degenerated and were lost. The failure of introduced varieties in India was mainly because these varieties were primarily bred to suit temperate long days of summer in Europe, whereas, potato in India is grown during short days of sub-tropical winters.
In view of the failure of exotic potato varieties and technologies under Indian agro-climatic conditions, a need was felt that potato cultivation in India cannot depend on exotic varieties and technologies and the country must have its own research and development program for potato.
A scheme for establishment of the Central Potato Research Institute was, therefore, drawn up in 1945 under the guidance of Sir Herbert Steward, the then Agricultural Advisor to the Government of India and the CPRI was established in 1949 at Patna.
Hills being the ideal location for producing and maintaining healthy seed and using wide potato genetic base through hybridization for breeding improved varieties, on the recommendations of an expert committee, the headquarters of CPRI was shifted in hills at Shimla in 1956.
During the period 1956 to 1979, a chain of regional research stations was established in different potato growing zones of the country to address local problems of potato cultivation. At present the institute has seven regional research stations located in different parts of the country.
These 7 regional research stations are:
1. CPRS, Patna
2. CPRS, Jalandhar
3. CPRS, Ooty
4. CPRS, Shillong
5. CPRS, Kufri/Fagu
6. CPRS, Modipuram
7. CPRS, Gwalior
Potato research at the institute is carried out in six disciplines, viz., Crop Improvement, Crop Production, Crop Protection, Crop Physiology and PHT, Seed Technology and Social Sciences.
Mandate of CPRI:
To undertake basic and strategic research for developing technologies to enhance productivity and utilisation of potato.
To produce disease-free basic seed of different notified varieties developed by the institute.
To act as national repository of scientific information relevant to potato.
To provide leadership and co-ordinate network research with state agricultural universities for generating location and variety-specific technologies and for solving area-specific problems of potato production.
To collaborate with national and international agencies in achieving the objectives.
To act as a centre for training in research methodologies and technology for up-grading scientific manpower in modern technologies for potato production.
To provide consultancy in potato research and development.
All India Coordinated Research Project on Potato [AICRP (Potato)]:
The Indian Council of Agricultural Research (ICAR) started AICRP on potato in 1970 with its headquarters at CPRI, Shimla, At present AICRP on potato has 22 centres located at regional research stations of CPRI/ICAR institutes and several State Agricultural Universities. (Table 34.10).
The mandates of AlCRP (Potato):
a. To undertake location specific research for identifying varieties and sustainable crop production technologies for enhancing productivity and utilization of potato.
b. To identify areas suitable for potato seed production.
c. To carry out multi-location trials on newly developed potato hybrids/TPS populations.
d. To evaluate agronomic practices including identification of remunerative potato based cropping systems in different regions.
e. To evaluate plant protection measures and post-harvest technologies aimed at increasing production and productivity of potato in the country as a whole.
f. To organize periodic workshops/group meetings to bring out recommendations based on the results of the coordinated trials to address the region specific problems in potato production.
Science Fair Project # 16. Varieties of Potato:
These are listed in Table 34.11:
