In Vitro Cloning Of Chrysanthemum

Chrysanthemum (Chrysanthemum morifolium R.) is one of the ornamental plants that are very popular in Indonesia. These flowers are cultivated by small farmers to big business on the land with an altitude of 600-1200 m above sea level. Small farmers cultivating chrysanthemums by applying simple technologies, while large businesses using modern technology-based agribusiness. Development of chrysanthemum is also a positive impact on the economy in rural areas, particularly the increased income of farmers and communities involved in its development.
In Indonesia, the demand for chrysanthemum increased 25% per year, even by the year 2003 the market demand increased 31.62%. Chrysanthemum exports to other countries like the Netherlands, Brunei, Singapore, Japan, and the UAE reached 1.44 million stalks (Plant Quarantine Station at Soekarno-Hatta 2003). High market demand that makes the chrysanthemum has a bright prospect to be developed both at present and the future (Ornamental Plant Research Center 2000).
According Rukmana and Mulyana (1997), chrysanthemum production business in Indonesia are faced with several obstacles, among others, dependence on seeds from other countries, like Netherlands, Germany, the United States, and Japan is expensive. In addition, if the plant will be copied to pay royalties of 10% of the selling price of each stem. The condition causes a high seed prices and lower profits farmers or entrepreneurs chrysanthemum plants.
Another problem is the degeneration of seeds, seed quality is decreasing with age as the parent plant, and the low quality of seeds produced. This is because the chrysanthemum shoots propagated by cuttings or seedlings (Rukmana and Mulyana 1997). To avoid or reduce the degeneration of seeds, manufacturers are required to update the parent plant on a periodic basis when the symptoms of degeneration became visible. Therefore, the development of varieties that have been produced by breeders of plants and application of appropriate propagation techniques are expected to resolve the issue.




Chrysanthemum is a flower plant ornamental shrubs form with another name or Flower Chrysanthemum Gold (Golden Flower) come from the plains of China. Yellow chrysanthemum comes from the plains of China, known as Chrysanthenum indicm (yellow), C. morifolium (purple and pink) and C. daisy (round, ponpon). In Japan the 4th century began to grow chrysanthemums, and 797 years djadikan chrysanthemum as a symbol of imperial Japan with the title Queen of the East. Chrysanthemum plants from China and Japan to spread to Europe and France in 1795. Year 1808 Mr. Chelsa develop Colvil from 8 chrysanthemum varieties in the UK. Types or varieties of modern chrysanthemum allegedly began in the 17th century. Chrysanthemum entered Indonesia in 1800. Since 1940, chrysanthemum developed commercially.
Botanical classification chrysanthemum plants are as follows: Divisions: Spermathophyta
Sub Division: Angiospermae Family: Asteraceae
Genus: Chrysanthemum Species: C. morifolium Ramat, C. indicum, C. etc. daisy.
Types and varieties of chrysanthemum plants in Indonesia are generally hybrids derived from the Netherlands, the United States and Japan. Chrysanthemums grown in Indonesia consists of: a) local Chrysanthemum (chrysanthemum ancient) Derived from abroad, but has a long and adapt in Indoenesia then considered local chrysanthemum. Its features include the nature of life in the neutral day life cycle and between 7-12 months in a single planting. Example C. maximum yellow flowers widely planted in Lembang and white flowers in Cipanas (Cianjur). b) introduction Chrysanthemum (chrysanthemum chrysanthemum modern or hybrid) for days her life is as short and annual plants. Examples of this chrysanthemum is C. indicum hybr. Dark Flamingo, C. i.hybr. Dolaroid, C. i. Hybr. Indianapolis (yellow flowers) Cossa, Clingo, Fleyer (white flowers), Alexandra Van Zaal (red flowers) and Pink Ping Pong (pink flowers). c) product Chrysanthemum Indonesia Ornamental Plants Research Center has released Cipanas varieties of chrysanthemum varieties of artificial Balithi Indonesia 27,108, 13.97, 27,177, 28.7 and 30.13A
Uses major chrysanthemum is a flower decoration. Another benefit is a traditional medicinal plant and insect toxin producing. As an ornamental flower, chrysanthemum in Indonesia is used as: a) Interest Identified by the figure of the pot small plants, 20-40 cm tall, flowering and suitable dense planted in pots or other containers polibag. Examples of mini chrysanthemum (small flower diameter) are varieties of Lilac Cindy (flower color purplefish ping), Pearl Cindy (pinkish white), Cindy White (white with white middle-green), Applause (bright yellow), Yellow Mandalay (all from the Netherlands). Krisan introduction of widely planted flowering as flower pots, there are 12 varieties potted chrysanthemums in Indonesia, which is the most planted varieties Delano (purple), Rage (red) and Time (yellow). b) Interest Identified by the figure of cut flowers to high short-sized, has a long flower stalk, varied sizes (small, medium and large), are generally planted in the field and the results can be used as cut flowers. Examples of cut flowers is very much including Inga, Improved funshine, Brides, Green peas, Great verhagen, Puma, Reagents, Cheetah, etc. Klondike.
Regional centers chrysanthemum producers include: Cipanas, Cisarua, Sukabumi, Lembang (West Java), Bandungan (Central Java), Brastagi (North Sumatra).
Chrysanthemum plants need adequate water, but do not withstand exposure to rain water. Therefore, for local high rainfall, planting in a plastic house building. 2) For flowering requires a longer light that is with the help of TL lamps and incandescent lamps. Addition of radiation is best middle of the night between 22.30-01.00 hours with 150 watt lamps for area 9 m2 and high mounted lamp 1.5 m above the ground. Period lighting installation is done until vegetative phase (2-8 weeks) to encourage the formation of flowers. 3) The best temperature as untukdaerah tropical Indonesia is between 20-26 degrees C. Tolerant of air temperature to keep growing is 17-30 degrees C. 4) chrysanthemum plants require high humidity to the early formation of roots of seedlings, cuttings required 90-95%. Young plants to maturity between 70 - 80%, balanced with adequate air circulation. 5) CO2 levels in the wild around 3000 ppm. CO2 levels are ideal for spur fotosistesa between 600-900 ppm. In chrysanthemum cultivation in closed buildings, such as plastic house, greenhouse, CO2 can be added, to reach the recommended levels.



Expand With Tissue Culture

Tissue culture is a method for isolating parts of plants such as protoplasm, cells, a group of cells, tissues and organs, and grow them in aseptic conditions, so that these parts can be propagated and regenerated into whole plants again.
The purpose tissue culture activities are mass propagation of plants is usually very slow with conventional methods in large quantities in a short time, but it acquired a virus-free plants, helping plants pemulian to accelerate the achievement of research goals in the regular plant vegetatively propagated.



The steps in Chrysanthemum Tissue Culture

Decision eksplan or source of eksplan chrysanthemum buds and nodes derived from the parent plants in a greenhouse chrysanthemum Balithi Germination planlet mountain and tissue culture laboratory Balithi mountain. Making Media MS media used for the chrysanthemum is a media mountain Balithi bud induction and multiplication media. Composition of media used for shoot induction was ½ MS + 0.5 IAA composition of the medium used for propagation was ½ MS + 0.1 IAA Preparing Eksplan the multiplication of tissue culture plants eksplan is an important factor determining success. The things that should be considered in selecting the material culture of the plant species, plant parts used, the surface morphology, growth environment, crop conditions, and the season take time. Generally the plant is used as a network of young eksplan current having a high regeneration.
Eksplan used in chrysanthemum plants is due to menginduks node aksilar buds. Aseptic culture of Chrysanthemum Sterilization Sterilization is an activity to remove contaminants attached organisms on the surface eksplan. The main purpose of this stage is to seek the aseptic culture and aksenik. Aksenik means free from undesirable microorganisms, whereas aseptic means free of microorganisms.

Selection And Preparation Plant Master

Before doing tissue culture plants for a first activity to be done is to choose parent plants to diperbanya. Plants. Selection to obtain clones, the desired clones. Clones that have different properties, unique, stable and uniform, then made the single stem plants and as a donor plant (eksplan materials) for propagation in vitro. Planlet (plants) results from the multiplication of in vitro and in greenhouse diaklimatisasi. After the plants to adapt to the greenhouse environment and then reproduced for the purposes of the parent plant that will produce the production plant.
It is very important for the environment should be tanamn parent heginis to get eksplan quality and cleaner for breeding in-vintro.
The work carried out in laminar space in order to avoid contaminants. Planting grouped by segment number. Each bottle filled 5 eksplan and repeated four times. Subsequent culture bottles incubated in growth room with 16-hour lighting fluorescent lamps under 40 watts, the temperature of 24-26 oC, and 60-80% humidity until eksplan grow planlet (plant tissue culture results had completed the plant parts including roots, stems, and leaves).


How to Sterilization For Chrysanthemum Plant

Taking eksplan already selected on the basis of durability Vigor, pest and disease, and the number of leaves from 4 to 5 pieces or
3 to 4 nodes.
• Cutting eksplan per node by reducing or cutting some leaves.
• Eksplan immersed in a solution Benlatte and Bactomicyn (fungicide and bactericide), each of 1 g/300 ml-aquades while jocok beaten for 30 minutes.
• eksplan rinse with water aquadest by 4 - 5 times.
• Next eksplan brought to laminar.
• Eksplan incorporated into a solution of 2 tetes/100 ml tween aquades with beaten-whipped for 5 minutes.
• Eksplan incorporated into Chlorox 0.5% solution for 5 minutes, shake-shake.
• Next eksplan inserted into Chlorox 1% solution for 3 minutes, shake-shake.
• Eksplan rinsed with distilled water as much as 5 to 6 times.



Eksplan Planting Planting Activities and Media

Eksplan planting culture in a bottle called inoculation. This activity is performed after eksplan sterilized, beginning with the cut surface eksplan. Eksplan next node in the form of two fruit grown in ½ MS medium + IAA 0.5 mg / l, while a top eksplan not need to be planted, just put it on the same medium as much as 3 units. Before closing with plastic wrap, clear plastic, and rubber, bottles that have been planted media first heated above the Bunsen flame. Next bottle labeled plant species and planting date. Eksplan who has taken to the dikulturkan incubation and allowed to grow.


Multiplication
Cutting or Multipikasi aims to reproduce propagule whereas, sub-culture is an attempt to replace the tissue culture media with new media, so that the nutritional needs for growth of callus can be fulfilled. The following is a description of the implementa-tion sub-culture techniques on solid media. From the first media, culture media was transferred to regerasi for about three weeks. After that, if we aim to reproduce or propagule multifikasi, then we do the transfer of new media and trunked plants if we do cuttings / cutting of chrysanthemum example we multiply by cutting perbuku. Or a couple of books depending on the point of growing leaves, which will become a new branch.
But usually done cuttig chrysanthemum growing stelah tanamn perfect and great. This is to memeudahkan multiplication, so it can memeprbanyak without limit.
Shoots in the laboratory propagation of tissue culture shoots were selected to obtain a healthy growth, good Vigor, and no symptoms deviation. Tunas elected then removed from the bottle carefully with a scalpel knife, then cut each segment / segments of the book from 1 to 4. Cutting is carried out in petridis using a knife culture. Plant part used is the top or sides 1, 2, 3, and 4. and stelah was incorporated into the culture media with media sub ½ MS + 0.5 IAA.
Root formation generally begins with the removal of indol acetic acid (IAA), produced shoots to the stems of plants that stimulate the formation of injuries to the root (Brenner et al. 1987).
After pomotongan, cuttings generally not sensitive to hormones. In this phase occurs dediferensiasi, the cells become competent and responsive to hormones. After that the active cells divide (are meristematic) Primordia formation followed by roots, root formation until the roots grow and develop (De Klerk et al. 1999). Auksin generally play an important role in the initiation of root formation. The role will be optimal if auksin environmental factors are also optimal.


Acclimatization

Acclimatization is the final activity tissue culture techniques. Acclimatization is the process of moving from planlet a controlled environment to uncontrolled environmental conditions, both temperature, light, and humidity. Acclimatization is to adapt the results of tissue culture plants to the new environment before it is planted and used as parent plants for the production and to determine the ability of adaptation of plants growing in environments that are less aseptic.
Environmental conditions change drastically, from the controlled environment is not controlled, the temperature is relatively stable to fluctuating temperature environments, from high to low humidity and fluctuating, and from low light to high light generally causes the plants susceptible to stress or stress, loss of water , wither, and die therefore, the process of acclimatization should be done in stages, as applied Winarto (2002) in carnations. Acclimatization will help crops adapt to changing environmental conditions such as temperature, humidity, and light intensity.

Acclimatization method is divided into 2, namely the direct method (direct) and indirect method (indirect).

Direct Method:
1. Preparing planlet in bottles that will diaklimatisasi and out planlet carefully from the bottle.
2. Cleaning the roots of plants from the agar-agar is still attached to the water.
3. Soaking the roots in a fungicide and bactericide solution for 5 minutes.
4. Planting crops in the media like charcoal that has been soaked husk.
5. Cover the tub with a transparent plastic sub 1 - 2 weeks.
6. 1 -2 weeks after the plastic is left open and the plants grow and thrive in the tub until the third week of acclimatization to fourth.
7. Later transferred to the plant-polibag small polibag until ready for planting in the field.

Indirect Method:
1. Preparing planlet in bottles that will diaklimatisasi and out planlet carefully from the bottle
2. Cut the plant right at the bottom of the third node and then immersion in a solution of fungicide and bactericide for 5 minutes.
3. Planting crops in the media like charcoal that has been soaked husk.
4. Cover the tub with a transparent plastic sub 1 - 2 weeks.


Planlet acclimatization on Greenhouse
Acclimatization is an important step in the process of tissue culture. This stage is often a critical point in the tissue culture technique applications. Acclimatization is required for plant tissue culture results generally have a thin wax coating and not yet well developed, the cells in the maximum-developed palisade, vascular tissue from roots to shoots are less developed, and stomata are often not functional, ie can not be closed at the time of evaporation high.


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