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biotechnology

Copies of perfection

ProClone is working to add quality and competitiveness to ornamental plant production in Brazil

MIGUEL BOYAYANKalanchoe Nursery beds in the greenhouse: the final cloning stageMIGUEL BOYAYAN

To reproduce the beauty of some flowers, to repeat that hue that only one orchid can manage to have at the peak of its flowering, these are the challenges that the cloning of ornamental plants has been pursuing for some years. The technique makes use of a minute piece of a cutting – more precisely the meristem, a grouping of cells of diameter 0,1 mm – in order to multiply flower samples without any genetic differentiating. Though in commercial use for more than twenty years, the technique is constantly being perfected. Whoever manages to use it with greater precision and at lower costs, producing perfect copies in large quantities, disease free and with unexpected combinations, earns s markets and a name. This is the objective that is in the sights of the company ProClone Biotecnologia, installed in the town of Holambra, in the Campinas region.

The company is in search of quality and competitiveness by increasing the production of clones of various plant species such as orchids, gerberas and arum lilies. Since 1997, with the support of FAPESP through the Technological Innovation in Small Companies Program (PIPE), ProClone, which produces 40,000 cuttings per month, has been technically preparing itself to take on the overseas market. Its exporting structure is in place, but “dormant” says the owner of the company, the biologist Monique Inês Segeren, who has associated herself to a Dutch company named Ceres Vitro in order to win over the foreign consumer. In this way, Monique will achieve one of her objectives that had led her to open the doors of her laboratory in 1989: to participate in the worldwide market of flowers and plants, which has a turnover, throughout its productivity chain, of close to US$ 100 billion per year. Of this total, the producers are responsible for an amount of approximately US$ 16 billion.

Holland is the principal worldwide importer and exporter of flowers and ornamental plants and serves as a base for the largest multinational companies in this sector. In Brazil, the production segment corresponds to 30% of the income, which reaches R$ 1.3 billion annually, the São Paulo State to be responsible for 70% of this value.

Only in Holambra there is more than R$ 273 million worth of flowers sold each year. This small municipality of some 10,000 inhabitants, situated at a distance of 155 kilometers from the state of São Paulo capital, had its origin in as farm, where a cooperative of Dutch farmers who had left Europe after World War II, was formed. During the last few decades, these farmers have dedicated themselves to the growing of flowers, becoming a national reference point in this sector.

In the attractive market of flower production, ProClone’s specialty is to clone seedlings created by improvement specialists, using an accelerated multiplication with all of the original characteristics of the preserved plants. This process, besides being defined as cloning is also called micro-propagation in vitro. The perfecting of this technique has been under development by the company since it began its participation in the PIPE program four years ago. Technical innovations have resulted in its own autoclaving equipment that carries out the sterilization and prepares the culture medium used in the containers for the seedling multiplication. After they are ready, the culture medium is distributed in a controlled manner to six hundred and fifty containers over a period of three hours. “In the manual process it would only be possible to fill up only twenty during the same time”. Besides the delay and margin of error, the manual method means greater energy and labor costs.

For now, the autoclaving equipment is installed at ProVitro, a joint venture formed between the flower entrepreneur and the company Ceres Vitro, which is being incubated at the High Technology Development Center Corporation (Ciatec in the Portuguese acronym) in Campinas. According to the entrepreneur, the sterilization of the culture medium with the autoclave will be done in the ProClone laboratory in Holambra, as soon as the expansion work has been concluded, and then a further two autoclave machines are installed.

Improved sterilization
The autoclave was designed and built by the engineer Sérgio Koseki using totally national equipment. It was patented by ProClone with the name Sterilization Equipment for Culture Medium and Automatic Distribution, and, according to Monique, there is no similar national model. A machine with similar functions exists in Germany, but the one developed by Brazilian technology has as a difference a helix, which makes the liquids more homogeneous and improves the sterilization.

For Monique, the fact that ProVitro is housed in an incubator is fundamental for the carrying out of the second phase of the project, the development of a complementary machine for the plant cloning process, which is already in the testing phase. We are talking about the plasma sterilizer of the company Valitech – another PIPE participating company at the Ciatec -, which uses hydrogen peroxide in the sterilization of plastic pots, a revolutionary process and with abated energy costs. In the words of the company researcher, Tadashi Shiosawa, the machine can sterilize two thousand plastic pots in an hour.

Other sterilization forms damage human health and the environment, as well as consuming lots of energy, explains the researcher. “Ethylene oxide is carcinogenic and causes environmental damage and the gamma rays radiation has restrictions as it a hazardous material”. The machine will also allow the re-use of the plastic pots after washing, which is not even done in Holland, added Monique.

With its new equipment, ProClone will have the conditions to offer a more elaborate and faster service to the first link in the plant and flower production chain, the so-called improver. This professional has specimens kept in greenhouses, which function as tools for the research of genetic variations and mutations. With them it is possible to create seedlings with their own distinct characteristics such as, for example, lilies with a different color or a banana resistant to a certain pest. In Monique’s judgment, these experiments fundamentally aim at the commercial use f the result. In the flower case improver shows his creation in exhibitions which work as showroom for plant and flower reproducers in search of new novelties.

Scale economy
As the improver has only some cuttings of the new product, it is necessary to carry out a multiplication so that the producer can place the plant on the market with speed and scale economy. Thus, the improver can pass the mother plant to a laboratory such as ProClone, where the multiplication of the seedlings via micro-propagation can take place. After tests with a group of plants to check quality, the lot to be multiplied on a large scale is chosen. The next stage remains in the same laboratory, or in the so called bio-factory. One seedlings is transformed into millions of cuttings, under controlled conditions, in a culture medium and with a reduced time frame. After this multiplication, the cuttings are delivered to the producer and only then come out of their pots to be planted in earth beds in the greenhouse, to grow, flower and consequently to be delivered to flower shops.

To guarantee that the seedling to be multiplied by this system is free of known illnesses, a quality test based on the Elisa method (Enzyme Linked Immunoabsorbent Assay) is carried out. The acceptance abroad of Brazilian cloned seedling goes through the Elisa test. It is an international standard method used to test the reactions that occur with cuttings in the face of known viruses and specific to each plant.

Certificate of origin
The consultant hired by ProClone, the researcher José Alberto Caram Souza Dias, of the Agronomy Institute of Campinas (IAC), adds that this test is the determining factor for obtaining a health certificate of origin, which proves the quality and the non-existence of virus and is an entrance guarantee of the seedling abroad. In his work at ProClone, Caram develops machines to carry out the test: they are an injector/washer and a dry blower for drying the microplates that hold ninety six cavities where the samples and reagents are placed, an operation that is repeated three times for each sample. The prices for these pieces of imported apparatus vary from US$ 2,000 to US$ 8,000. Caram estimated that the two machines adapted by him will cost less than R$ 1,000.

Mother plant evasion
Greater technical capability in the production of ornamental plants in Brazil could reverse a roll back that is at the minimum worrying: the evasion of Brazilian other plants, in which the most flagrant case is that of various species of native orchids that are being reproduced abroad and re-exported back to Brazil. According to Monique, an imported orchid seedling costs R$ 4, while it is possible to reproduce it here at R$ 10 per lot of fifty.

The flower entrepreneur adds that today there are four groups (genres) of orchids with excellent commercial acceptance – Phalaenopsis, Dendrobium, Oncidium, known in Brazil as golden rain, and Paphiopedilum, known as the little shoe – and two others also with a great demand for reproduction:C atleya and Laelia, genres appreciated by orchid lovers who reproduce and sell these plants in exhibitions and flower markets. “The Brazilian collection is widely spread, but the orchid is a plant highly dependent on the laboratory”, emphasized Monique.

ProClone is also accelerating its multiplication of arum lily (Zantedeschia), a genre with forty species of varied color, which has a growing demand on the international market. This plant is imported from Holland, but its origin is South Africa. Other plants that make up the shelves of ProClone are the Gerbera (Gerbera), the Kalanchoe (Kalanchoe), as well as ferns such as the ferns Dicksonia, Polypodium and Philodendron. ProClone is also carrying out the reproduction in vitro of varieties of Anthurium (Anthurium) developed and produced by the IAC.

Monique’s strategy is quite clear: clone and develop seedlings that the market desires and beat the competitor and foresee consumer wishes. To achieve it, she is innovating in the equipment production, looking for niches in the market and associating herself with technology and investment partners. The company has six employees and five scholarship students. The financial support of development agencies in her research has been part of the routine since the start of the company’s activities through the Human Resources Training Program for Strategic Activities (Rhae) of the Ministry of Science and Technology (MCT).

Investment in production
While she was waiting for the FAPESP project to be approved, Monique fulfilled a work program of eight months in Kenya, where she managed a laboratory with more than one hundred employees of the Ceres Vitro company. This experience was important in solidifying the entrepreneurial links with her partner Hendrikus Petrus Schouten for the formation of ProVitro. He, as well as Ceres Vitro, is also the owner of Ceres International with its headquarters in Holland.

The eye of the Dutch partner passes at least four times per year over the new and embryonic company, for which they have just purchased an area of seven hectares in the Holambra region. There, as soon as the entrepreneur identifies the correct moment to invest, construction of a bio-factory will begin that is going to produce cloned cuttings of various species – on a large scale – with good prospects for exporting. The estimated cost for this undertaking is of R$ 1.5 million. For now Monique says that her partner, on his visits to Brazil, is prospecting the ground and getting to know the partner producers, who in their hothouses will be reproducing the improved cuttings of ProClone.

The Project
Development of Seedling Quality Control in Laboratories Allied to Biotechnology (nº 99/11514-6); Modality Small Business Innovation Research (PIPE); Coordinator Monique Inês Segeren – ProClone; Investment R$ 263.000,00

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