AK Srivastava, LC Diengdoh, R Rai, and TK Bag
Central Potato Research Station, 5th Mile, Upper Shillong, Shillong – 793 009
The potato is readily amenable to tissue culture manipulations. Many techniques have been developed for growing potato in tissue culture. As a result tissue culturally potato multiplication has successfully been incorporated in high quality potato seed production programme. Meristem culture in combination with thermotherapy and chemotherapy is now routinely utilised to obtain pathogen free potato microplants which are serially multiplied through nodal cuttings. The potato microplants can be multiplied through nodal cuttings or can be utilised for microtuber production. The microplants and microtubers are planted in polyhouses to obtain minitubers from them. These minitubers are then multiplied twice in field to increase their numbers and sizes. Aeroponics system has also been utilised for soilless production of minitubers. It has advantage over other micropropagation methods in having higher multiplication rate and greater control on the size of tubers harvested.
Keywords Tissue culture, potato seed
Over the past 50 years the application of cell and tissue culture techniques has been most conspicuous in potato than any other crop species. The first successful establishment of tissue culture from potato tubers was reported as early as in 1951 (Steward and Caplin, 1951), and since then in vitro cultures of potato were developed from different plant parts such as petioles, ovaries, anthers, stems, roots, and shoot tips (Bajaj, 1987). Due to its high amenability to in vitro manipulations, a range of techniques had been perfected in this crop over the years. These techniques are of differing degree of complexity forming a complete spectrum of technologies. While some of these technologies have been applied to improved potato production by mean of micropropagation and pathogen elimination, others are still being refined and improved. The use of in vitro technique for virus elimination (meristem culture) and clonal mass propagation (micropropagation) is the most prominent application in potato. In vitro produced disease free potato clones combined with conventional multiplication methods have become an integral part of seed production in many countries (Naik and Sarkar, 2000). Although some of the basic techniques like in vitro selection, microspore and protoplast regeneration and somatic fusion have been available since the late 1970s, the challenge is to make this procedure reproducible, universal and economic so that these can be integrated into practical potato improvement programmes.
Pathogen elimination through meristem culture
As early as in 1952, Morel and Martin observed that many viruses were unable to infect the apical and/or auxiliary meristem of a growing plant, and that of a virus free plant could be produced if a small (0.1 -0.3 mm) explants of meristematic tissue was propagated in vitro. Since more than 30 viruses are known to infect potatoes, this technique found its immediate application for virus elimination. In the absence of chemical control of viral diseases, meristem culture technique is the only effective method available to date that eliminates virus infection from the systematically infected potato cultivars. In general, larger the size of the meristem, more the chances of its survival in vitro, whereas, smaller the size of the meristem, lesser are its chances of being virus-infected (Mellor and Stace-Smith, 1987). Although theoretically it is possible to eliminate viruses from potato plants following meristem culture alone, this procedure is almost always combined with thermotherapy and/or chemotherapy to increase the likelihood of obtaining virus free plants in vitro (Khurana and Sane, 1998). Thermotherapy involves growing whole plants or in vitro cultures at high temperature close to the threshold of normal plant growth. The effectiveness of thermotherapy is due to disruption in the synthesis of viral ssRNA and/or dsRNA, production or activity of virus encoded movement proteins and coat proteins. The movement proteins are involved in cell to cell movement of viruses through plasmodesmata and long distance movement through plant vascular system (Martin and Postman, 1999). For most plant cultivars, thermotherapy is usually done at 37oC, however, the exact temperature and length of treatment varies with the virus and heat tolerance of the plant. In contrary to thermotherapy, antiviral chemicals are used in chemotherapy to inhibit or interfere with virus replication or movement in plant tissues. These chemicals can either be sprayed on growing plants or incorporated into tissue culture media. Meristem culture in combination with thermotherapy and in vitro ribavirin therapy at low concentration has been used successfully to eradicate viruses from infected potato cultivars (Sanchez et al., 1991). The virus free clones of selected potato cultivars are maintained and multiplied in vitro for specialized utilization in potato seed production, international exchange and other activities. The protocol for potato meristem culture consists of i) selection and testing of apparently healthy plants from the field, ii) establishment of in vitro cultures and iii) meristem culture.
Selection and testing of plants
- Select apparently healthy plants from the field or sample tubers.
- Test these plant/tubers for freedom from viruses using ELISA.
- If no plants/tuber is found free from all the viruses then one has to resort to meristem culture.
- Select a plant/tuber that is infected with minimum (one or two) viruses for use in meristem culture.
Establishment of in vitro cultures
- From infected plant: When starting with plant, excise nodal segments from the third and fourth node from the stem apex with a scalpel. Each nodal cutting should be 1-2 cm long, and the leaves should be detached. Such single node cuttings (SNCs) are used to initiate in vitro cultures.
- From infected tubers: Treat the freshly harvested tubers with 0.2% Bavistin for 15 min and dry them. If not required for immediate use, the tubers can be stored at 40C for about 1 year. For immediate use, allow the tubers to sprout at 240C. Since most cultivars are dormant after harvest, sprouting will initiate at least after 2 months. Harvest sprouts measuring about 2-3 cm long.
- In the laminar flow clean air work station, surface sterilize the nodal segments/sprouts for 10-15 minutes in 20% of commercial grade of sodium hypochlorite solution (4% w/v available chlorine), rinse in sterile distilled water for three times. Trim both ends of the explants by a scalpel and place the explants into individual culture tubes (25 x 150 mm) each containing 13 ml of semi-solid nutrient medium.
- The culture is based on MS (Murasighe and Skoog,1962) basal nutrients supplemented with D-calcium pantothenate (2mg/l), Gibberellic acid (0.1 mg/l), alpha- naphthalene acetic acid (0.01 mg/l) and 30g/l sucrose. The medium was solidified with 7.0 g/l agar.
- Incubate the cultures under a 16h photoperiod from cool white fluorescent lights (approx. 50-60 mmol/sq m/s light intensity) at 240C.
- Allow the explants to grow up to 6-8 nodes/stem stage, and then subculture through SNCs on fresh medium under the same culture conditions. Shoot cultures can be maintained and multiplied in vitro by sub culturing on fresh medium every 3 weeks.
Thermotherapy: It can be given to in vitro cultures or tubes prior to meristem culture. This is done as follows
- Place 7 day old cultures in a BOD incubator at 370C under a continuous photoperiod (approx. 20 mmol/sq m/s light intensity), and incubate for three weeks.
- Treat infected tubers with GA3 (2 mg/l) and allow them to sprout at 370C under dark till 8-10cm long sprouts are formed.
- After thermotherapy, meristems are dissected from in vitro plantlets and/or sprouts by the method described below.
Meristem excision and culture
- Excise meristem (terminal as well as axillary) from thermo-treated in vitro plantlets under laminar flow cabinet using a stereoscopic zoom microscope, scalpel and needle. Protective leaves on the buds are removed carefully using needle. Use a drop of sterile distilled water to avoid meristem desiccation during excision.
- Trim the meristematic dome plus one set of leaf primordial with a scalpel to 0.2-0.3 mm.
- In case of sprouts, surface sterilization of sprouts using 20% of commercial grade of sodium hypochlorite solution is essential before dissecting meristems.
- Place the excised meristems on semi solid meristem culture medium in a culture tube (1 meristem/culture tube), and incubate the cultures under a 16 h photoperiod (approx. 50-60 mmol/sq m/s light intensity) at 240C.
- The meristem culture medium is based on MS basal nutrients supplemented with 2 mg/l D-calcium pantothenate, 0.1 mg/l GA3, 0.01 mg/l NAA and 30g/l sucrose, and solidified with 6.0 g/l agar.
- It takes about 5-6 months for meristems to grow into full plantlets (mericlones). At this stage sub-culture the plantlets, maintain their clonal identity.
- Test meristem derived plantlets for presence or absence of viruses by ELISA and/or ISEM.
- Multiply and maintain virus-negative counterparts of meristem-derived clones by shoot cultures in vitro as described above.
Micropropagation allows large scale asexual multiplication of pathogen free tested potato cultivars. At an interval of every 21 days of sub culturing, minimum 3 nodal cuttings are obtained from a single microplant. Therefore, theoretically 315 (43 million) microplants can be obtained from a single virus free mericlone in a year. Various techniques have been developed for producing large number of microplants on nutrient medium under aseptic conditions. Nodal segment culture in which axillary and terminal buds grow into new plants is predominantly used for initial shoot multiplication. The method involves culturing of nodal explants of disease free microplants on semisolid (agarified) or liquid culture medium. Considerable research has been done on the nutritional, hormonal and physical aspect of the culture media and their effects on explants growth. Murasighe and Skoog medium is most widely used for potato micropropagation. Semisolid medium is used for initial nodal segment propagation; however liquid medium fosters higher growth rate of potato micro shoots (Rosell et al., 1987). In vitro derived microplants are used as i) explants source for the production of microtubers in vitro, ii) direct transplants in the greenhouse for the production of minitubers, iii) mother plants for further in vitro multiplication through single node cuttings (SNCs) and iv) source material for production of synthetic seed.
Microtuber production in vitro
Microtubers are miniature tubers developed under tuber inducing conditions in vitro. These small tubers are particularly convenient for handling, storage and distribution. Unlike micro propagated plantlets, they do not require time consuming hardening periods in greenhouses, and may be adapted easily to large scale planting in the field. Many protocols have been developed to induce microtubers in potato. Explants can be nodal cuttings, excised stolons, micro shoot cuttings or whole microplants.
The harvested micro tubers are dormant, and therefore, required to be stored at 5-60C for 3-4 months before planting on nursery beds or in the field. In order to avoid weight loss of microtubers during storage, microtubers are greened before harvesting by incubating 50-65 days-old induction cultures under 16h photoperiod at 240C for 10-15 days (Naik and Sarkar, 1997). Greening improves the storage of microtubers in terms of reduced biomass loss due to shrinkage and better sprout emergence. Thickening or suberizing the periderm of microtubers during greening is mainly responsible for making them more tolerant to evaporative water loss. Moreover, glycoalkaloids produced during greening protect the microtubers from bacterial and/or fungal damage.
Microtuber production technology involves i) initial multiplication of virus-free plantlets on semi-solid medium, ii) mass multiplication of in vitro plantlets in liquid medium, iii) production of microtubers, iv) harvesting and storage and v) field planting. These are depicted in plate 1.
- Multiply disease-free stock plants (obtained from meristem culture) through nodal cuttings on semisolid MS medium in culture tubes (25 x150 mm). To economise on space, tubes and other inputs, use ordinary cane sugar in place of sucrose and inoculate 3 nodal cuttings in each tube.
- To maintain varietal identity edible colours @ 25mg/l can be mixed in the medium before autoclaving.
- Incubate the cultures under a 16h photoperiod from cool white fluorescent lights (approx. 50-60 mmol/ sq m/s light intensity) at 240C.
Mass micropropagation in liquid medium
- When large number of plants is produced in initial multiplication, liquid cultures are initiated for mass micropropagation.
- Initiate liquid propagation cultures in 250 ml Erlenmeyer flasks or magenta box, pour 20ml liquid propagation medium (composition same as in semisolid medium except agar) and autoclave.
- Inoculate 10-12 stem segments (each having 3-4 nodes) obtained from six 21-day-old plantlets in each flask/box
- Incubate the liquid cultures under the same cultural conditions as in semisolid propagation.
- In about 3 weeks all axillary buds grow into full plants and fill the container.
- After 21 days of incubation, decant the liquid propagation medium from the Erlenmeyer flask or magenta box under aseptic conditions of a laminar flow workstation, and pour in 40ml of microtuber induction medium. The microtuber induction medium is based on MS basal nutrients supplemented with 10mg/l N6-benzyladenine (BA), 500mg/l chlorocholine chloride (CCC) and 80g/l sucrose (commercial sugar).
- Incubate these induction cultures under complete darkness at 200C. Microtubers start developing epigeally at the terminal or axillary ends of the shoots within 8-10 days and they are ready for harvesting after 60-90 days depending upon genotype. In general, 15-20 micro tubers with an average weight of 100-150mg are produced in each flask or magenta box.
Harvesting and storage
- Before harvesting, green the microtubers in vitro by incubating the induction cultures under a 16h photoperiod (approx. 30 mmol/ sq m/s light intensity) from cool white fluorescent lights at 240C for 10-15 days.
- Hand-harvest the green microtubers in plastic trays. Avoid damaging the microtubers during harvesting.
- Wash the harvested microtubers in running tap water to remove adhering constituents of the medium. Treat the harvested microtubers with 0.2% Bavistin for 10 minutes and allow them to dry in the dark at 200C for 2 days.
- Pack the dried microtubers in perforated polythene bags and store at 50C in a refrigerator for 4-5 months under dark for breaking dormancy.
- After 3-4 months of storage, the sprouted microtubers are ready for field planting.
- Plant the sprouted microtubers (1 microtuber/plastic bag) in perforated plastic bags (8x4cm) filled with 1:1:1 mixture of farmyard manure, sand and soil, and grow for 3 weeks in the greenhouse.
- Transplant the established plantlets into the field by removing the plastic bags without disturbing the root-soil mass.
- Irrigate the plots by furrow irrigation throughout the growing period according to evapo-transpiration requirements to avoid drought stress during crop growth and grow the crop following recommended package of practices. At maturity harvest normal sized tubers.
Aeroponics in potato
Aeroponics culture refers to soil less culture for producing minitubers. It involves spraying plant roots with a fine mist of a complete nutrient solution. Aeroponic systems feature the roots of the plant growing in lightproof, sealed box or container where they are continuously or intermittently misted with nutrient solution. This solution is continuously recirculated through the system. The top portion of the plant is exposed to the open air and a light source (either artificial light or natural sunlight). It does not need any excess area for aeroponic based healthy seed production. Only one percent of conventional water usage is required which is basically recycled water. It is the ideal technology for cost-effective production of quality seed in the present era. Advantages of this system are i) tropical states which do not have isolated and virus-free potato growing areas can also produce quality seed ii) early supply of nucleus seed to commercial growers by reducing the field exposure time iii) improved tuber quality and reducing the load of degenerative diseases iv) utilize the resources and trained manpower round the year and v) vertical growth and reduction in pressure on land.
Soil less production techniques, such as Nutrient Film Technique (NFT) and aeroponics have been successfully employed in tuber production with good prospects for certified seed production (Boersig and Wagner, 1988). Aeroponic system for seed potato production has also been successfully established in Korea (Kim et al. 1997, 1999) under tropical and sub tropical conditions. Farran and Mingo-Castel (2006) reported that plant density and harvesting intervals influence the minituber production in aeroponic system. They also reported that the field performance of aeroponically produced minitubers was comparable to the minitubers produced from other techniques. There is a tremendous scope to increase healthy seed production vertically by adopting aeroponic technology where increase in multiplication rate from 5:1 to 50:1 can be achieved. Aeroponic system has comparatively higher multiplication rate as compared to conventional micropropagation system. Harvesting in aeroponics is convenient and allows a greater size control by sequential harvesting. It has added advantage such as solution recirculation, a limited use of water and is safe from soil borne diseases.
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