Practical 3.2 : Totipotency and plant tissue culture Objective: To demonstrate the totipotency of plant cells.Introduction: Totipotency Totipotency is the ability of a cell, for example ovum, to give rise to unlike cells and thus to develop into or generate a new organism or part when the embryo develops and the cells looses totipotency. In the experiment of cloning by nuclear transfer of adult cells, we have to 'reprogram' the karyoplast so that we could have a cell with full totipotency. Otherwise, the reconstructed embryo would not be able to develop into a new being. Brassica rapa Brassica rapa are also known as turnips. It’s a biennial herb with swollen tuberous white-fleshed taproot, lacking a neck; leaves light to medium green, hairy or bristly, stalked, lyrate-pinnatifid, 30–50 cm long, stem-leaves sometimes glaucous with clasping base; flowers bright yellow, sepals spreading: petals 6–10 mm long, those in anthesis close together and commonly overtopping the unopened buds; outer 2 stamens curved outwards at base and much shorter than inner stamens; fruit 4–6.5 cm long, with long tapering beak, on divaricate-ascending pedicels 3.2–6.5 cm long; seeds blackish or reddish-brown, 1.5–2 mm in diameter. Brassica rapa subsp. fungi. Roots are also grown for feeding to livestock during fall and winter. photoperiod. sulfur dioxide. Most common white-fleshed varieties are: 'Purple Top White Globe' and 'White Egg'. or home-garden crop. green or purplish-red. and weeds (Duke. frost. truck crop.. rapa. Roots are eaten raw or cooked as a vegetable. laterite. 'Seven Top' is grown in South for useof greens. Brassica rapa subsp. grown as a fodder crop. native to Asia. Field mustard is a weed or ruderal in much of Europe.. disease.p. as general farm crop. p. 'Purple Top White Globe' is recommended for tropics.Varieties may have white or yellow flesh. Pekinensis Group—pe-tsai or "Chinese cabbage". cultivated for its tuberous taproot. and outside crown may be white. mycobacteria. widely grown in the South for greens and salad. bacteria.) Janchen (B. 1978). (2n = 20) Turnips are one of the most commonly grown and. is reported to. has larger reddishbrown seeds and non-tuberous taproot. . high pH.widely adapted root crops. sometimes escapes as a weed. and Ruvo Group—ruvo kale. Yellow-fleshed turnips include 'Golden Ball' or 'Orange Jelly'. low pH. sylvestris (L. Brassica rapa subsp.). campestris L. turnip. tolerate aluminum. 'Shogoin' is a white-skinned. turnip. virus. 'Amber or Yellow Globe' and 'Yellow Aberdeen'. Rapifera Group—turnip. Eurosiberian. and Mediterranean Centers of Diversity. oleifera DC. Perviridis Group—spinach mustard. or cvs thereof. Turnip rape. Reported from the China-Japan. Terrell (1977) divides Brassica rapa into the following groups: Chinensis Group—pak-choi. and tops as potherb like spinach. white-fleshed Japanese variety. smog. 2 to 7. 0.1). and linolenic acids. But turnips occasionally suspected of poisoning bovines. The stems and leaves are said to be a remedy for cancer. 92. 168 mg Ca. resistant to frost and mild freezes.6 to 27. 1979). The root when boiled with lard is used for breast tumors.04 mg thiamine. Brassica rapa is reported to tolerate annual precipitation of 3. 1978. Further on in 1972. Watt and Merril reported that per 100 g. 49 mg Na. 0.07 mg riboflavin.6 mg Fe.9 g fiber. In 1963. Turnips do well in deep.6 g ash.6 g total carbohydrate. a trace of carotene equivalent.18 mg riboflavin. sheep. linoleic.5 mg Fe. the leaf contain 23 calories.5 g H2O.0 g fiber. 1. 0.4°C (mean of 75 cases = 10.7 mg niacin.7 g ash.2) (Duke. 1. 0. annual temperature of 3. The powdered seed is said to be a folk remedy for cancer. 0. 1330 mg -carotene equivalent.8 (mean of 66 cases = 6. 2. and pigs.7). sandy loams are used for early markets roots and greens. Turnip is basically a cool climate crop. 6. 39 mg Ca. and 36 mg ascorbic acid. the root contain 30 calories. 30 mg P. 1. Temperatures below 10°C would cause bolting.0 dm (mean of 75 cases = 9. and pH of 4. 0.9 g protein.8.2 g fat. 0. . Turnips are grown as a spring or fall crop throughout the United States.6 mg niacin. 268 mg K.2 g fat. 91. highly fertile soil with pH 5. 0. Ranging from Boreal Moist to Rain through Tropical Thorn to Moist Forest Life Zones. 52 mg P.5 to 41. Short growing season makes them very adaptable as a catch crop. friable. 4. Wu Leung reported that per 100 g.7 g H2O. while a salve derived from the flowers is said to help skin cancer. 0.Turnips are also used for medical purposes. Seed oil contains large amount of erucic. 420 mg K. 0. 78 mg Na. 0.10 mg thiamine. 0.5–6.6 g total carbohydrate.0 g protein. and 47 mg ascorbic acid. in well-drained soils. They should not be left in the ground where temperatures near freezing occur.6 m wide nor more than 2 m deep to prevent heating at the center. in milder areas they may be left in field until desired.Turnip’s seed sown thinly in spring. Seedlings then thinned to stand 5–15 cm apart in rows 0.1–2.5–6. peas or grass crop (Reed. or they may be used as a catch crop after early vegetables. well-prepared seedbeds where weed control will not be difficult. Good rotation helps to control diseases. They may be stored in pits or piles. Flavour and texture are not improved by storage. Cultivate shallowly for weed control. Turnips may be intercropped with corn.3–0. Seed may be broadcast on fertile.5 cm in diameter. washed. It is not advisable to grow turnips after a root crop. . turnips are pulled. as 450–675 kg/ha of 4–12–4. Alternate layers of straw and soil are used as covering for pit storage. Add lime to soil to correct pH to 5. When turnips are seeded as a fall crop following a crop that has been well fertilized.5–3 m in the pile. Turnip greens may be harvested when plants are young and tender. beans. 1976). and for topped turnips when 7. Topped turnips for the general market are sold by the bushel or the hundredweight. wooden chutes are inserted at intervals of 2. For good aeration. summer or fall in drills at seed rate of 1. A ditch is dug around the base of pile for water runoff. For early spring market.2 kg/ha. Best grown after clover. Roots may be harvested in 45–80 days. and as such they are shade-tolerant. Piles should not be more than 2. and marketed.8.9 m apart. Only light applications of fertilizer are justified. They are harvested for bunching when 5 cm in diameter. no additional fertilizer may be necessary. tied in bunches. their tops left on. Storage temperature in a cellar or in a cold storage room should remain between 0° and 1. .000 MT. the residues remaining for potential energy conversion. crates or small piles laid on earth cellar floors are satisfactory. Good yields are 12. However. According to the phytomass files. The national consumption for both turnips and rutabagas (these are usually reported together) is ca 200. Turnip crop in 1969 was ca 60. annual productivity ranges from 4 to 11 MT/ha. Indian studies showed DM yields of 530–1.000 MT.000 kg/ha. 820–2.5°C. Duke reports fresh turnip yields of 65 MT/ha in 1978.260 kg/ha after 38 days with 61– 191 kg extractable protein. Turnips and turnip greens are available all year. these figures do not represent true yields since it is difficult to determine what proportion of a crop is sold topped. and plots were cropped continuously (perhaps impractical. Small quantities of turnips may be stored in a cool cellar and covered with moistened clean sand to keep them from drying out.For indoor storage. bunched or fed to livestock.5 tons/bunched or 25 tons/topped per hectare in the United States. if not impossible). If this much were available in 45 days. with a relative humidity of 90–95%.000 MT. and the oil from such seeds is being considered for energy purposes. not counting quantities used for animal feed. the 1971 consumption was ca 50. Seed yields in Minnesota and Canada run over 1. with peak production in October and November. DM yields might run 6–16 MT/ha with ca 800–2.090 kg/ha after 52 days with 90–265 kg extractable protein.000 kg/ha per year. small plastic tray . campestris. cling film. Alternaria brassicae and A. Materials: Apparatus: Seeds of Brassica rapa. by various insects. but 2 or 3 hives are sufficient to increase pollination and to insure good seed set. Report of Practical Aim: Problem Statement: To investigate the role of totipotency in plant tissue culture. Viruses isolated from turnips include: Beet mild yellowing. scissors. Bacterium aroideae and Erwinia carotovora. paper towels. Turnip aphid. juncea. root maggot and flea beetles are the most injurious insect pests. agar. and B. in New Zealand. Nematodes attacking turnips include: Belonolaimus longicaudatus. short-necked test tubes. distilled water Damp sponge. 250 ml beaker.Cross pollination. 16–17 colonies of bees/ha are used. 400 m. brassicicola. In USSR. is necessary for good seed production. B. weighing scale. permanent pen. Does the cells of plant tissue culture able to develop into a new complete plant? Hypothesis: The explant will continue to grow provided if the conditions are favourable and there is no contamination. Ditylenchus dipsaci and Trichodorus christiei. Beet ringspot and Curly top. Isolation of varieties necessary for pure seed production. Other fungi attacking turnips include: Albugo candids. Should be wellisolated from all other forms of B. Turnips may be parasitized by Orobanche cernua. napus. Clubroot (Plasmodiophora brassicae) and Black rot are the most serious diseases. in England at least 900 m. glass rod. or attacked by the following bacteria: Agrobacterium tumefaciens. the tray was covered with transparent cling film and placed in a warm. The tubes were placed under the light bank. The tops just below the shoot apex were cut off the seedlings with a sharp pair of scissors.5 g of agar powder was measured and added to 250 cm3 of distilled water. The agar was poured about 2 cm depth into several short-necked test tube while it still in molten state. The tubes were covered with cling film and labelled. Some seeds of Brassica rapa were sprinkled onto a damp sponge placed in a plastic tray. The progress of explants were observed and recorded daily for 10 days. 2. light place to germinate. 6. 3. These were called explants. The agar was boiled and stirred with a glass rod until the agar dissolved.Procedures: 1. Then. 5. 7. Results: . It is allowed to cool and solidify. 4. 2. The cut end of each explant was carefully pushed into the agar. They are ready to culture when the seedlings start to unfold their cotyledons. 9 Plants remain wilted.9 cm 5 5 New leaves are becoming more obvious. colour of leaves becoming more paler and yellowish. 0. Discussion: . size of leaves do not have much change.7 cm Number of leaves 4 2 Good conditions. 0. 7 Plant remains wilted. broad leaves and 2 leaves joined together.Days 1 Explant conditions Plant in good condition. no root growth is detected and colour of leaves turn paler.8 cm 4 4 New leaves start to appear on the areal part. height of the explant and number of leaves in explant for 10 days. the size of leaves increase.8 cm 4 3 Good conditions. growth can be seen but measurement cannot be taken due to bad condition. medium sizes of leaves. Agar surface covered with white patches. 0. colour of leaves becoming more paler and yellowish. Height 0. 10 Leaves become transparent Table 1 shows the condition of explant. size of leaves do not have much change.9 cm 5 6 Wilted. 8 Plants remain wilted. 0. Analysis of the data The experiment is conducted to investigate the totipotency of Brassica rapa explant. As the explant grow. Besides. a short-necked test tube is used instead of beaker or other container. the growth of roots. the tube is covered with cling film to avoid the air from contaminating the explant.9 cm up until day 5 of the experiment. The number of leaves and the height of the stem increases. These require a careful observation as a slight change in plant will give significant effect in results. they can differentiate into . causing observations and measurement of further growth is halted. This is due to the presence of bacteria and microorganism in the air. the tubes cannot be open back. The length of stem is also being measured in this experiment. Opening the film would expose the explant to the contaminated air. number of leaves and number of explants should be measured to show the totipotency in Brassica rapa. The explant has 5 leaves and the height increases to 0. Based on Table 1. The experiment did not show positive result due to responsible errors and limitations that will be discussed further on. As the tube is covered. On the sixth day onwards. number of explants and also the length of stem. As plant cells are totipotent. This is because it is easier to put the explant into the agar medium without actually contaminating the agar and the explant. number of leaves. agar is used as the medium for the explant to grow. the explant is growing. For the experiment. The totipotency of the explant can be measured by their growth of roots. After the explant is planted into the agar. The agar supplies all the essential mineral ions that contribute to the growth of explant. the explant wilt and eventually die. In order to measure the stem length. As in Brassica rapa. the use of scalpel might already cause the explant to damage. Before handling the glassware. Be extra cautious when handling sharp utensils such as scissors and scalpel. Besides. Precautionary steps and risk factors Laboratory coat is worn throughout the experiment to avoid spillage when handling the molten agar that may stain clothes and solidify. the explant demonstrate totipotency in the growth of roots. This causes the explant and agar exposed to air and being contaminated. This is possibly causing the explant to immerse into the agar and causing the cotyledons to actually touch the agar. Localise each portion of the tissue culture procedure in a specified place in the laboratory. be careful when handling glassware such as short-necked test tubes as they are fragile. Wear oven gloves as the agar is hot. the cling film needs to be removed from the tube. Besides. late in the experiment. the measurement used to measure totipotency includes the measuring of length of stem. But. This might also lead to contamination. hands should be washed despite using sterilised apparatus. An assembly-line arrangement of work areas facilitates all operations and enhances cleanliness. The plant is held to tight and this causes the stem to swell. the experiment is not conducted in the Laminar flow. most of my group members do not wash their hands. the agar used might not fully solidify. So. stem and new leaves. the explant is exposed to the air and possibly already contaminated by breath. . white patches can be observed ressembling the presence of bacteria. That is why. Despite that. In my experiment. In fact.all type of cells. finally. use glassware that has only been used for tissue culture and not other experiments. then rinse several times with tap water and. Toxic metal ions absorbed on glassware can be especially troublesome. Ensure that the cotyledons of the explant do not touch the agar when it is planted into the agar to avoid contamination. Only high-purity water is used in tissue culture procedures. Wash glassware with laboratory detergent. Double glass distilled water or deionized water from an ionexchanger are acceptable. Water should not be stored. The essence of aseptic technique is the exclusion of invading microorganisms during experimental procedures.First of all. Common sterilants are ethyl alcohol and/or chlorox with an added surfactant. If sterile tissues are available. . Brassica rapa need to be healthy and actively growing. rinse with purified water. but used immediately. Surface sterilization procedures specific to the Brassica rapa are employed. Concentration of sterilants and exposure time are determined empirically. then the exclusion of microorganisms is accomplished by using sterile instruments and culture media concurrently with standard bacteriological transfer procedures to avoid extraneous contamination. So. Besides. Moreover. the explant is too small to observe the changes in physical structure. This will cause the agar to be contaminated by microorganisms and air particles. the sample is left at the laboratory without any supervision. This causes the agar and the explants to be exposed to air which also leads to contamination. One of them is the experiment is conducted in an area exposed to air. Any environmental changes will affect the growth the explant in terms of temperature. the brassica is grown randomly. In addition. there may be some defects on the plant. light intensity and humidity. . For example. students have to use the explant given even if there is any damage to the plant that could have contaminated or not healthy anymore. in order to avoid re-opening of the film. This can be overcome by conducting the experiment in the Laminar flow where the air inside it is clean and constantly flowing. So.Limitation and modifications Existence of limitations is unavoidable and thus can affect the accuracy of the results. In fact. The defective may form when the plant is placed exposed to the environment when the students were choosing the plant. the labelling should be made before the tube is covered with the cling film. ensure that the procedures are followed step by step. the amount of Brassica provided is limited. the tubes are open once they are covered. Therefore. Besides that. 2008.htm http://www.com/uk/orc/bin/9780199282616/ch02. Hodder Education Ann Fullick.pdf http://www.ableweb.oup. Edexcel AS Biology. The results are compared with the explant that is grown with their shoot apex and cotyledons together.edu/newcrop/duke_energy/brassica_rapa.hort. Edexcel Biology for AS. Hypothesis is not accepted for this experiment. This is because the cotyledons supply the plant with nutrients. Absence of cotyledons will cause the plant to depend for nutrients from the agar which is limited.html http://www. References: C J Clegg. So.org/volumes/vol-11/9-mineo. Pearson Company http://library. Conclusion: The explant in this experiment is unable to show the characteristic of totipotency due to the present of certain limitations.thinkquest.Further investigation Tissue culture can also be made by growing the shoot apex alone with the stem into the agar.pdf . as the plant grows.purdue. the cotyledons also grow which then provide continuous supply of nutrients to the plant. The cotyledons are removed first before the explant is placed into the agar. The explants with the shoot apex and cotyledons grown together grow successfully compared to the explant grown with the shoot apex alone. 2008.org/C0122429/glossary/totipotency.