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Rose, a symbol of love, affection, inspiration and spirituality, is one of the leading cut flower in the world floriculture trade. No other flower is as universally loved or has a brilliant history as rose. Roses have been admired for their beauty, color and fragrance since the dawn of civilization. Some rose species have fragrance due to the presence of volatile compounds in their petals and used to extract essential oils. These good quality essential oils are highly valuable for cosmetics and pharmaceutical industries. New cultivars development utilizes seeds while cuttings and budding are commercial methods of propagation. These methods of propagation are not sufficient to produce large number of good quality and high yielding plants to fulfill growing demand because these methods have slow multiplication rate, attack of different insect pests, diseases and unfavorable weather conditions.
To produce healthy and disease free plants of roses round the year, micropropagation is a viable technique which can help in production of approximately 4,00,000 plants from a single rose plant per annum. Now, the work has been started on the micropropagation of Rosa centifolia by direct and indirect methods at Institute of Horticultural Sciences, University of Agriculture, Faisalabad and Arid Agriculture University, Rawalpindi.
Micropropagation methods of regeneration are different from the conventional propagation methods in a number of ways. In the micropropagation way, artificial growth medium is used instead of soil, light, temperature and humidity are adjusted according to plant requirements and any plant part can be used. Healthy and disease-free plants are produced without any season effect and many plants can be produced from a single plant part, haploid plants can be produced, plant breeding cycle can be shortened and can be used to produce mutations in plants.
For different micropropagation techniques (meristem culture, axillary bud culture, root culture, leaf culture, anther culture) any plant parts (axillary bud, root, leaf, anther etc.) can be used because every cell has the ability to regenerate (totepotency). Explants are first excised, sterilized with alcohol for 5 minutes followed by three washings with double distilled autoclaved water and then with sodium hypochloride (bleach) for 5 minutes again followed by three washings of double distilled autoclaved water. Now, the explants are sterilized and ready to be cultured on nutrient medium i.e. MS (Murashige and Skoog, 1962) medium, which is made up of micronutrients, macronutrients, vitamins, iron and different concentrations of growth regulators according to requirements of the plant.
After adding all components, pH of the solution should be adjusted at 5.7 and agar (gelling agent) is added in the solution after heating in the oven and filled in the test tubes. All the test tubes, after covering with polythene and wrapping with rubber band, are sterilized in autoclave at 1210C and 15 Psi pressure for 20 minutes and placed in the growth room. Before start, cultures inoculation room is sterilized by UV radiations for 30 minutes and then explants are cultured in the test tubes in vertical position. Test tubes are again covered, wrapped and placed in growth room at 2520C under white tube lights of 2500 Lux and almost 70 per cent humidity. After 3 weeks, shoots will develop from the nodes, of which proper sized shoots are transferred in the rooting medium with different concentrations of growth regulators for root development. According to our results, BA 3.0 mg L 1 NAA 0.5 mg L 1 proved best for shoot formation while addition of NAA 1.5 mg L 1 BA 0.5 mg L 1 in MS medium was best for root formation. After root development, young newly developed plants are acclimatized. During acclimatization, first plants are transferred into small pots filled with locally available fully decomposed compost containing 1-2% nitrogen, 1 per cent phosphorus, 1 per cent potassium and approximately 25 per cent organic matter. Plants are transferred in small pots after removing media from their roots because microorganisms can attack on roots due to sugar in the media. Pots are covered with polythene sheet to maintain the humidity because young plants require more humidity as in the test tubes. After 4-5 days, a cut is made on one side of sheet and gradually removed after 2 weeks and uncovered plants are retained in the growth room followed by 1 week placement in the lab. After a few days, plants are transferred to greenhouse in big pots and can be transplanted or marketed.
Process of micropropagation of roses
Different problems also arises during micropropagation of roses and their control is very important otherwise whole effort will be useless. Major problem is browning in which medium and explant become brown and finally dies. This is due to phenolic compounds released from the cut portions of the woody plants. These phenolic compounds block the uptake of nutrients from media by the explants and cause death of the explant. This problem can be controlled by adding charcoal in the medium; treatment of explants with citric acid and ascorbic acid and also by placing explants in the running water for 45 minutes. Another problem is contamination that occurs due to microorganisms. Contamination may be due to medium or explant. Contamination of medium is due to improper wrapping of test tubes and of explant is due to improper sterilization and culturing. This is a major source of contamination and causes maximum loss of plant tissue culture and micropropagated plants. In order to control contamination, media should be properly sterilized in the autoclave and then placed in the growth room. All test tubes should be properly wrapped. Before culturing all tools and laminar airflow cabinet should be sterilized with alcohol and inoculation room with the UV radiation.
There are different direct and indirect techniques of micropropagation. In direct techniques, shoots are developed from the explant tissue without callus formation e.g. meristem culture. In this technique small, (0.2-0.5 mm) pieces of meristem are used as explant. This technique is used for virus eradication because meristem has faster cell division than virus multiplication. In seed culture, seeds are cultured on nutrient medium to develop shoots and roots. In indirect techniques, first callus is developed from the explant and from callus, shoots and roots are developed e.g. leaf culture. In this technique, small leaf segments having mid rib are used to culture and produce callus, which further develops into shoots and roots. In anther culture, anthers (male part of a flower) are used as explant to produce haploid plants used in the breeding programme. For root culture, root segments are used as explant. In cell culture, only cell can be used as explant after separating from the tissue. Ovary and flower petals can also be used to develop in vitro plants.
Micropropagated plants are healthier, disease free and produce higher yield of better quality more oil contents, rapid growth rate and aesthetic value. Genetic variability can also be produced in micropropagated plants. For fulfilling rapidly increasing demand for centifolia roses, micropropagated plants are need of the day. In order to boost micropropagation business in the country, following suggestions should be taken into account.
Developing a market for micropropagated plants, creating awareness among the people, more research to develop protocols of micropropagation for different species establishing new plant tissue culture laboratories, more short courses to produce skilled manpower, government incentives to start micropropagation business and develop cooperation among private and government sectors for enhancing production and export of good quality micropropagated plants.
The authors are associated with the Institute of Horticultural Sciences, University of Agriculture, Faisalabad, Pakistan.