This action might not be possible to undo. Are you sure you want to continue?
CHAPTER 3 3 BIOREACTOR STUDIES ON MICROPROPAGATION OF DENDROBIUM, AN ORCHID 3.1 STERILIZATION OF PODS The time required for mature seed formation of Dendrobium is 100 -140 days. The precise time for harvesting the immature seed is temperature and particularly species dependant. Therefore, roughly 70 days old green undehisced pods were harvested because the seeds were approximately progressed to ½ to 1/3 in their development which was found suitable as explants for their culture in the preliminary experiment. The pods were cleaned thoroughly under tap water followed by washing with 5% Tween 20 for 10 minutes and finally with distilled water. They were surface sterilized with an aqueous solution of 0.1% mercuric chloride for 15 minutes and were subsequently rinsed 3 times in autoclaved distilled water. The segments are then washed in 70% ethanol for 2 minutes. Then the explants were removed from the sterilizing solution and rinsed thoroughly for two times with sterile double distilled water. 3.2 INITIATION OF PODS
After surface sterilization, the capsules were taken in a sterile petridish containing filter paper to absorb the surface water. Sterilized pods were transferred aseptically to sterilized glass plate under the laminar flow hood.The capsules were cut longitudinally and powdery mass of yellowish seeds were inoculated on the slant surface of 0.8 % agar solidified Knudson C(KnC) nutrient medium (1951).supplemented with NAA (α–naphthalene acetic acid) at different concentrations and combinations with 15 -25 % coconut water. pH of the media was adjusted to 5.4 and autoclaved at 15 kpa
There was a highly significant variation in percent seed germination among different media has been observed in required days to seed germination.The present findings have similarity with their results. ornamental. The seeds were aseptically inoculated into test tubes of culture medium. Present studies have demonstrated that immature seeds obtained from green pod of Dendrobium a pharmaceutically valuable. Ismat (1982) and Hoque (1993) found that required days for seed germination of Dendrobium in MS medium is 51 and 55 days respectively. can be germinated asymbiotically in vitro for rapid micropropagation. . epiphytic forest orchid having horticultural importance.The rate of survival of plantlets after the transfer from culture vessel to natural condition was 98-99%.51 for 15 min at 121oC.Knudson C (Kn C) medium (1951) containing 0. Cultures were then incubated at dark condition for 30 days at 20±1oC and later cultures were kept on the same media without any subculture upto 45 days at 20±1oC under 16 hr photo period under the cool white fluorescent light of average 2500 lux (cool white fluorescent tube light 40 W GE).1 mg l-1 NAA and 15% coconut water (CW) was found most effective for high percentage (80-90%) of seed germination and seedling development.This method can be exploited for the rapid propagation and conservation of Dendrobium.
3 INDUCTION OF PLB’s (protocorm like bodies) A procedure was developed for protocorm-like bodies (PLBs) formation and proliferation and subsequently plant regeneration from seeds of pod of Dendrobium.These protocorms proliferated and formed a cluster of upto a dozen protocorms.they formed a spherule –like body with rhizoids at the base these structures are protocorm like bodies.1mg/lNAA+15% CW KnC+0.5mg/l NAA+25% CW KnC+ No supplement 3.52 Table 3.5mg/l NAA+15% CWl KnC +0.1mg/lNAA+25 % CW KnC+0.1 mg/l NAA KnC +0. instead of developing into a leafy shoot. When cultured.1 MEDIUM CODE FOR DENDROBIUM PROTOCORMS MEDIUM CODE (KC-KC8) MEDIUM CODE KC1 KC2 KC3 KC4 KC5 KC6 KC7 KC8 KC MEDIUM DETAILS KnC+15% CW KnC+25%CW KnC +0. . These protocorm mass were cut into three to six pieces and planted them on fresh Knudson C Medium.5 mg/l NAA KnC +0. Each piece formed additional protocorms by the activity of the superficial cells.
in the inner space ethylene gas accumulates. however. But some orchids are not amenable to this approach.53 From each piece upto 12 protocorms were produced in less than a month. If chopping were stopped each protocorm developed into a full plantlet on the original medium. This process of protocorm multiplication could be repeated indefinitely by regularly chopping the protocom mass and planting the pieces on fresh medium. which has a . and/or they do not develop perfectly under the special conditions of stress caused by the in vitro growing methods applied till now. For example. The spreading of the traditional method is also limited by the fact that certain species and varieties are still difficult to cultivate. which increases the price of reproduction material considerably. bulbs. each piece again gives rise to several protocorms. etc.. Cymbidium. generally. rapid clonal propagation were achieved by inducing adventitious buds. In this manner. For example. Each protocorm divides to yield several (up to a dozen. The technique is widely used in orchid industry for micropropagation of both sympodial as well as monopodial orchids. When protocorms are left as such without chopping. excised shoot-tips of most orchids. produce round bodies similar to the protocorms formed by embryo during seed germination. etc. each protocorm gives rise to a complete plantlet on the same medium. Cattleya. and thus their vegetative growth. Traditional micropropagation is a labour intensive plant biotechnological method. The closing system of the traditional cultivation vessels prevents external infection. In many plant species. the cultivation media using agar-agar or other materials to form a gel do not serve properly the plants absorption of nutrients. these bodies are also called protocorms. 3-5) protocorms. Each protocorm may be cut into 4-6 pieces and subcultured.. e.g. This approach can yield over 106 orchid plants in one year from a single shoot-tip. Vanda. the protocorms can be multiplied indefinitely. protocorms.
But when leaf. A shoot bud arising anywhere other than a leaf axil or the shoot-tip is called adventitious shoot bud. Each bulblet is separated and gives rise to a complete plant.8000 plants can be obtained from a single shoot-tip in about 4 months. some adventitious shoot buds would be formed in most plant species. the basal half of each bulb scale is cut into about 6 pieces. The outer bulb scales show high regeneration capacity. Each 1 mm shoot-tip is cut into 20 pieces. stem or other explants of plants are cultured in vitro. adventitious shoot buds are used for micropropagation. Owing to the cumulated effect of the above facts. e. Adventitious shoot buds are produced in vivo in many plant species. a number of plant species cannot be competitively micropropagated in vitro by the traditional methods. fragmented shoot-tips have been used to induce adventitious shoot buds. In grapes. Begonia leaves. etc. In Begonia. It may be pointed out that even during axillary bud proliferation. etc. and each fragment may produce multiple shoots.g.54 hormonal effect and promotes biological aging. these are often used for convention. and each scale produces an average of 18 bulbs lets in 4-6 weeks. Generally. Micropropagation of lily (Lilium longiflorum) is done by inducing bulb formation on cultured pieces of bulb scales... upto. A similar approach is applicable to Narcissus. . In many species. while inner scales show progressively lower potential. which is a very high multiplication rate. Phlox roots. up to 1014 plants can be obtained in a year from a single 7 mm x 7 mm leaf explant. propagation of species like Begonia. a very large number of adventitious shoot buds develop. Saintpaulia. Thus a single bulb scale may yield about 100 bulblets.
undesirable/brownish tissues were removed from the PLB’S and were taken to the culture bottles containing autoclaved semi-solid media having the same combinations as that for the culture initiation. . All this work was done with extreme care and inside the laminar flow hood to avoid any possible chance of contamination.55 3.4 ESTABLISHMENT OF CULTURES After approximately 9-10 days of inoculation. the initiated PLB’S were taken out the test tube. After 21. Multiple shoots/cluster were transferred from the culture bottle to a sterile glass plate using flamed sterilized forceps. medium adhered to the PLB’S were removed. 3.25 days of incubation with a clean and sterilized forcep under the laminar flow hood. these cultures were then transferred to jars containing fresh medium. the proliferation of seeds were seen in some seeds (explants).5 MULTIPLICATION OF SHOOTS BY REPEATED SUBCULTURING IN MULTIPLICATION MEDIA The preparation and sterilization steps for the medium. These shoots were transferred to the multiplication media. When the explants attain proliferation. instruments and chamber were repeated as before. the brown leaves were removed from the primary shoots and sectioned into one shoot piece after removing from the primary shoots. Then the bottles were placed in the culture room under the standard conditions of temperature (25± 2°C) for 16/8 hrs of day/night break under the cool white fluorescent light of average 2500 lux (cool white fluorescent tube light 40 W GE).
56 These culture bottles were then incubated in the growth room. which is different from the shoot multiplication medium. The time required for in vitro rooting of shoots may vary from 10 – 15 days. With the help of sterile forceps elongated shoots up to 1-2 cm in length were placed into the rooting medium. Rapid clonal propagation was achieved by subsequent induction of protocorm-like bodies (PLBs) and its conversion to shoots. The culture bottles were then capped and placed in the growth room under the same condition.6 ROOTING OF THE SHOOTS Axillary shoots developed in cultures in the presence of cytokinin generally lack roots. especially in its hormonal composition. A low salt medium is found satisfactory for rooting of shoots in large number of plant species. 3. .7 ROOTING PROTOCOL In the laminar flow. These steps were repeated every 25-30 days for the next sub-culturing. Table 3. To obtain full plants the shoots must be transferred to a rooting medium. 3.2 SOIL MIXTURE CODE FOR PLANTS (HM1 – HM3) HM1 HM2 HM3 SOIL SOIL׃VERMICOMPOST(2׃1) * SOIL׃VERMICOMPOST(4׃1) * *parameter measured in v/v. under sterile conditions the Klin film and cap was removed from the culture bottles in sterile condition (laminar flow hood) and with the help of sterile forceps the multiplied shoots were removed from the medium and placed on the sterile glass plate.
g. multistage bioreactors and immobilized cell bioreactors. which has provisions for (i) (ii) (iii) (iv) aeration.8 MULTIPLICATION OF PLB’S IN SUSPENSION CULTURE Use of liquid medium in suspension cultures allows easy and extensive scaling up by employing bioreactors. stirring to achieve medium and cell mixing. e. except the last one are commonly called stirred tank reactors since the vessel has a device for stirring. continuous bioreactors.. which provides possibility for exploiting resources by large scale culture of PLBs. I L to 1000 L. contamination control and replacement of used medium and/or used medium and cells. generally of a large volume. A bioreactor is a culture vessel. PLBs were suspended in liquid medium and growth kinetics was analyzed: PLBs in liquid culture system show a potential for rapid growth and high metabolite synthesis. all bioreactors. The bioreactors used for culture of plant cells may be of following four types: (1) (2) (3) (4) batch bioreactors.57 3. although a limited scaling up can be achieved by using larger culture flasks usually 250 ml flasks having 50 ml medium are used). .
Hyponex medium was found to be suitable for conversion of PLBs into plantlets and 83% of PLBs transformed into plantlets on this medium. Lindemann and Hyponex media. Continuous immersion cultures (air lift column and air lift-balloon bioreactor). Protocorm-like bodies (PLBs) formed on leaf segments in vitro were used as explants for bioreactor cultures. A temporary immersion culture with charcoal filter attached was most suitable for PLB culture. Knudson C.0 volume of air per volume of medium min^−1 (vvm) yielded similar levels of biomass production.9 MULTIPLICATION OF PLB’S IN BIOREACTOR Protocorm-like bodies (PLBs) formed on seeds in vitro were used as explants for bioreactor cultures. The feasibility of using PLBs for .58 3. rate of proliferation. Vacin and Went. The feasibility of using PLBs for large-scale micropropagation was evaluated for scaled-up liquid cultures in bioreactors. PLBs grown in bioreactors were cultured on solid Murashige and Skoog. Bioreactor-cultures have several advantages compared with agarbased cultures. and temporary immersion cultures (with or without charcoal filter attached) were used for the culture of PLB sections. Aeration in a bioreactor at 0.5 or 2. and temporary immersion cultures (with or without charcoal filter attached) were used for the culture of PLB sections.000 PLBs were harvested from 20 g of inoculum ( 1000 PLB sections) in 2 l Hyponex medium after 8 weeks of incubation. Micropropagation in bioreactors for optimal plant production will depend on a better understanding of plant responses to signals from the microenvironment and on specific culture manipulation to control the morphogenesis of plants in liquid cultures. as well as aeration and medium circulation. the filtration of the medium and the scalingup of the cultures. and regeneration. with a better control of the contact of the plant tissue with the culture medium. About 18. and optimal nutrient and growth regulator supply. Continuous immersion cultures (air lift column and air lift-balloon bioreactor).
tissues and organs. An internal loop bioreactor was used for asparagus. Bioreactor-cultures have several advantages compared with agarbased cultures. mostly guided by an automatic control unit. utilizing liquid media to avoid intensive manual handling. and optimal nutrient and growth regulator supply. Bioreactors are special devices developed for the effective in vitro cultivation of cells. the filtration of the medium and the scalingup of the cultures. A simple glass bubble-column bioreactor for the proliferation of ornamental and vegetable crop species resulted in biomass increase of 3 to 6-fold in 3-4 weeks. technical means which can program plant growth. The most important components of the equipment are the following: cultivation containers made of glass. Micropropagation bioreactors form one of the newest subgroup of the so-called fito-bioreactors equipped with artificial illumination. Their common characteristic is that they are apt for the . as well as aeration and medium circulation. on the basis of this. Various types of bioreactors with gas-sparged mixing are suitable for the production of clusters of buds. celery and cucumber embryogenic cultures. rate of proliferation. and regeneration. Micropropagation in bioreactors for optimal plant production will depend on a better understanding of plant responses to signals from the microenvironment and on specific culture manipulation to control the morphogenesis of plants in liquid cultures. Bioreactors provide a rapid and efficient plant propagation system for many agricultural and forestry species.59 large-scale micropropagation was evaluated for scaled-up liquid cultures in bioreactors. probes sensing the development of the cultivated plants and. Large-scale liquid cultures have been used for micropropagation through organogenesis or somatic embryogenesis pathways. with a better control of the contact of the plant tissue with the culture medium. meristems or protocorms.
The bioreactor has the following components: a timer. by the regulation of the chemical. Hence. . an inner container. humidifier. bioreactor cultures are superior to conventional solid or liquid cultures. shoots. quality and quantity of light. embryos and roots.60 programmed direction of the optimum growth of vegetative tissues. a small electronic pump. seeds. two plastic jars connected with a silicon tubing to allow medium to circulate between the two. Lateral buds on the flower-stalks induced protocorm-like bodies (PLBs) of orchid were cultured in liquid medium with air-driven periodic immersion (API) bioreactors. However. the API bioreactors can be used in large quantity of Orchid culture and may even be applied to the commercial micropropagation of orchid and other ornamental plant. for example buds. physico-chemical és physical factors of the inner and outer space (cultivation media. Our results indicated that mass proliferation and plantlet differentiation rates of Orchid PLBs in API bioreactor are affected by many factors. and the inner gas space) influencing in vitro morphogenesis.
This action might not be possible to undo. Are you sure you want to continue?
We've moved you to where you read on your other device.
Get the full title to continue reading from where you left off, or restart the preview.