Whether it's increasing yields, cutting growth time of plants, increasing their resistance to disease or preserving genetic resources, scientists say tissue culture could be an important answer …

Scientists at Jawaharlal Nehru TAKE A small part of a shoot tip and put it in a test tube. Then add some chemicals that will nurture the tissue and lo and behold, the part grows into a full plant.

This may sound like witchcraft, but it is precisely what tissue culture technology is all about. And given its vast promise, not only is the Department of Biotechnology (DBT) giving it high priority, but industries in the biotech boomtowns of Hyderabad, Bangalore and Madras are also excited.

Because tissue culture enables scientists to duplicate a specific plant, cell or organism, it opens up a vast vista of cloning specific plants or micro-organisms that have elite characteristics such as high yield or resistance to disease. Tissue culture also enables new plants to be produced bypassing the normal reproductive cycle -- production of seeds, for instance.

V Jagannathan, former head of the biochemicals division of the National Chemical Laboratory (NCL) in Pune, sees vast prospects for tissue culture, both in agriculture and forestry. "Tree breeding takes 50 to 100 years. But better varieties of trees can be developed much faster with tissue culture, and, using tissue culture and genetic engineering techniques, India can wipe out plant viruses," says Jagannathan.

The DBT has approved about 15 units for the production of tissue-cultured plants both for the domestic market and for export. Domestic consumption of such plants is about two million now, while exports amount to about six million. When all the approved units begin production, the total will rise to about 100 million plants and export earnings are estimated at about Rs 50 crore a year, against a total investment in tissue culture production facilities of about Rs 55 crore. Companies intend to produce foliage, flowering and tropical and temperate fruit plants and plantation crops in collaboration with firms from such countries as France, Italy, Belgium, UK and the Netherlands (See box). A V Thomas & Co, a leading tissue culture company, for instance, is planning exports of tissue-cultured orchids in a big way.

DBT has also invested heavily in the development of tissue culture technology focussing on trees for fuel and fodder, bamboos, oil palm and on other plantation crops such as cardamom. Research institutes such as NCL, Bhabha Atomic Research Centre (BARC), Indian Institute of Science, Tata Institute of Fundamental Research (TIFR), Indian Agricultural Research Institute (IARI) and Jawaharlal Nehru University (JNU) are involved in basic and applied research to develop tissue culture techniques for a variety of plants. Yet another area of research promoted by DBT and the Ministry of Environment and Forests is to use the new technology to protect India's endangered plants. (See box)

Indian scientists have been at the forefront of research in tissue culture. Even though the possibility of taking a single cell and converting it into a full plant had been predicted as far back as in 1902, it was only in 1958 that Western scientists were able to get an embryo -- the starting point of a new plant like a seed -- from a mass of cultured carrot cells. In 1964, S Guha and Satish Maheshwari of the University of Delhi reported in Nature that pollen grains in cultured anthers of Datura innoxia developed into embryos. This was a major development in tissue culture.
Simultaneously, a team of scientists at TIFR headed by M M Johri has been working on cellular differentiation -- the process by which one cell becomes the leaf and another becomes the stem -- and their findings have become textbook knowledge. "Tissue culture work is still a bit like witchcraft, explains Johri. "There is a lot of hit-and-miss because the basic mechanisms are still not understood well. That is what we are trying to do."

In l990, NCL scientists A F Mascarenhas, R S Nadgauda and V A Parasharami reported flowering in tissue-cultured bamboo shoots grown from seedlings. Given that bamboo of the varieties grown by NCL scientists would normally not have flowered before 30 years, a commentator wrote in Nature (Vol No) that the NCL achievement opened up "endless possibilities for the High Emperor of all the Grasses".

Lab-industry links
But, as yet, there are few links in India between tissue culture industry and research labs. The Madras-based confectionery manufacturer, E I D Parry, did get its initial technology for virus-free, tissue cultured sugarcane from NCL. But, says Parry research manager J Subramani, "We had to change the culture technique and storage systems." And, P Nanthakumar, chief manager of tissue culture at Southern Petrochemical Industries Ltd (SPIC), adds, "We at SPIC developed the technology in-house. It took us two years. If you are willing to invest in time and money, you can develop your own technology. On the other hand, if you want to start commercial production from tomorrow, you will have to import technology."

Many scientists object to importing plantlets, saying it leaves Indian scientific expertise in the cold. Usha Rao of the University of Delhi favours the government acting as an intermediary between Indian and foreign companies and then deciding who should get what work.

Officials at SPIC and Parry agree the main problem with Indian technology is that it holds good for only a few hundred or thousand plantlets, but fails when it involves millions. "As a result, industry has to invest in its own R&D or import technology," says Thomas.

C Sivarama Reddy, executive director of Bio-Tissue Labs in Hyderabad, who worked on tissue culture while associated with Andhra Pradesh Agricultural University, is even more blunt: "Companies go to scientists, but their technology does not work. On paper they claim that they can produce millions of plants, but only a few develop in reality."
Indian scientists have been at the forefront of research in tissue culture. Even though the possibility of taking a single cell and converting it into a full plant had been predicted as far back as in 1902, it was only in 1958 that Western scientists were able to get an embryo -- the starting point of a new plant like a seed -- from a mass of cultured carrot cells. In 1964, S Guha and Satish Maheshwari of the University of Delhi reported in Nature that pollen grains in cultured anthers of Datura innoxia developed into embryos. This was a major development in tissue culture.
Simultaneously, a team of scientists at TIFR headed by M M Johri has been working on cellular differentiation -- the process by which one cell becomes the leaf and another becomes the stem -- and their findings have become textbook knowledge. "Tissue culture work is still a bit like witchcraft, explains Johri. "There is a lot of hit-and-miss because the basic mechanisms are still not understood well. That is what we are trying to do."
In l990, NCL scientists A F Mascarenhas, R S Nadgauda and V A Parasharami reported flowering in tissue-cultured bamboo shoots grown from seedlings. Given that bamboo of the varieties grown by NCL scientists would normally not have flowered before 30 years, a commentator wrote in Nature (Vol No) that the NCL achievement opened up "endless possibilities for the High Emperor of all the Grasses".

Indian scientists have been at the forefront of research in tissue culture. Even though the possibility of taking a single cell and converting it into a full plant had been predicted as far back as in 1902, it was only in 1958 that Western scientists were able to get an embryo -- the starting point of a new plant like a seed -- from a mass of cultured carrot cells. In 1964, S Guha and Satish Maheshwari of the University of Delhi reported in Nature that pollen grains in cultured anthers of Datura innoxia developed into embryos. This was a major development in tissue culture.

Simultaneously, a team of scientists at TIFR headed by M M Johri has been working on cellular differentiation -- the process by which one cell becomes the leaf and another becomes the stem -- and their findings have become textbook knowledge. "Tissue culture work is still a bit like witchcraft, explains Johri. "There is a lot of hit-and-miss because the basic mechanisms are still not understood well. That is what we are trying to do."

In l990, NCL scientists A F Mascarenhas, R S Nadgauda and V A Parasharami reported flowering in tissue-cultured bamboo shoots grown from seedlings. Given that bamboo of the varieties grown by NCL scientists would normally not have flowered before 30 years, a commentator wrote in Nature (Vol No) that the NCL achievement opened up "endless possibilities for the High Emperor of all the Grasses".

Lab-industry links
But, as yet, there are few links in India between tissue culture industry and research labs. The Madras-based confectionery manufacturer, E I D Parry, did get its initial technology for virus-free, tissue cultured sugarcane from NCL. But, says Parry research manager J Subramani, "We had to change the culture technique and storage systems." And, P Nanthakumar, chief manager of tissue culture at Southern Petrochemical Industries Ltd (SPIC), adds, "We at SPIC developed the technology in-house. It took us two years. If you are willing to invest in time and money, you can develop your own technology. On the other hand, if you want to start commercial production from tomorrow, you will have to import technology."

Many scientists object to importing plantlets, saying it leaves Indian scientific expertise in the cold. Usha Rao of the University of Delhi favours the government acting as an intermediary between Indian and foreign companies and then deciding who should get what work.

Officials at SPIC and Parry agree the main problem with Indian technology is that it holds good for only a few hundred or thousand plantlets, but fails when it involves millions. "As a result, industry has to invest in its own R&D or import technology," says Thomas.

C Sivarama Reddy, executive director of Bio-Tissue Labs in Hyderabad, who worked on tissue culture while associated with Andhra Pradesh Agricultural University, is even more blunt: "Companies go to scientists, but their technology does not work. On paper they claim that they can produce millions of plants, but only a few develop in reality."

Indian scientists have been at the forefront of research in tissue culture. Even though the possibility of taking a single cell and converting it into a full plant had been predicted as far back as in 1902, it was only in 1958 that Western scientists were able to get an embryo -- the starting point of a new plant like a seed -- from a mass of cultured carrot cells. In 1964, S Guha and Satish Maheshwari of the University of Delhi reported in Nature that pollen grains in cultured anthers of Datura innoxia developed into embryos. This was a major development in tissue culture.

Simultaneously, a team of scientists at TIFR headed by M M Johri has been working on cellular differentiation -- the process by which one cell becomes the leaf and another becomes the stem -- and their findings have become textbook knowledge. "Tissue culture work is still a bit like witchcraft, explains Johri. "There is a lot of hit-and-miss because the basic mechanisms are still not understood well. That is what we are trying to do."

In l990, NCL scientists A F Mascarenhas, R S Nadgauda and V A Parasharami reported flowering in tissue-cultured bamboo shoots grown from seedlings. Given that bamboo of the varieties grown by NCL scientists would normally not have flowered before 30 years, a commentator wrote in Nature (Vol No) that the NCL achievement opened up "endless possibilities for the High Emperor of all the Grasses".

Hyderabad-based Unicorn Biotek, considered at one time tying up with NCL for their cardamom technology, but backed out later. "There were several people working in cardamom, so we opted out. Dealing with the government has its own hassles," said Unicorn Biotek managing director Amul Sanghani.


Jagannathan contends India missed the bus in tissue culture even though it had an early scientific lead because India continued with lab research, while industrialised countries focussed on applications research. "The only option now," he says, "is to intensify our research and increase interaction between our industries and research labs."


But this means university research will first have to be streamlined, which may prove onerous. Says Rao in exasperation, "For everything, we have to fight -- to get finances, to get recognition. Scientists should be working and not looking after trivial matters." Echoing her sentiments was IARI's S K Raina, "In India, bureaucratic delays act as deterrents. It is private industry that has shown that big business can come out of tissue culture."


Others like Kamaljit Singh of Indo-Australian Flora Pvt Ltd in New Delhi see no role for the government in popularising tissue culture. "Government can help by staying out; it should sleep," says Singh. But Bio-Tissue Labs' Sivarama Reddy wants more encouragement from export promotion councils because "export enquiries are rarely forwarded to us and international trade fairs are very expensive for fledgling businesses".


DBT has taken up tissue culture research right from its inception in 1986. Its support was given initially to research on bamboo propagation, on which Delhi University scientists had been working for long, and to research on tissue-cultured oil palms and coconuts. The work on bamboo is at an advanced stage and tissue-cultured plants are being field-tested in various parts of the country. Work on oil palm and coconut was taken up at the behest of the government's technology mission on oilseeds as the country was importing large quantities of edible oils.

Most of the coconut acreage in India is under the traditional tall varieties, which yield 30-50 nuts per tree every year. Elites amongst traditional varieties yield 150-200 nuts per tree per year and hybrid varieties can give as much as 350.

DBT also decided to launch a major research project on oil palm. Field plantations of test tube oil palm were already existing in Malaysia and some other countries. The work on oil palm was entrusted to BARC in Bombay and the Central Plantation Crops Research Institute (CPCRI) in Kasaragod, Kerala. Work on coconut was assigned to four institutes: CPCRI, NCL, Tamil Nadu Agricultural University (TNAU) in Coimbatore and JNU in New Delhi. DBT was so confident in its work, it planned to start mass production of tissue-cultured coconut by 1995.

Slow progress
However, when the two projects wound up in 1989-90, progress in both coconut and oil palm research was found to be slow. A revaluation followed and only BARC, which by then had got 60 tissue-cultured plantlets, was permitted to continue work on oil palm for three more years and only NCL was allowed to go ahead with its work on coconuts. JNU's Shipra Mukherjee explained, "Coconuts have proved to be a particularly recalcitrant plant" and coconut cells cultured in the laboratory failed to grow roots and shoots. "The project was stopped because there was not enough success," Mukherjee added. "Coconut contains several inhibiting substances that leach out into the culture and prevent its growth. New techniques will be needed to regulate these inhibitors."

In 1988-89, DBT started a cardamom tissue culture project because Indian export of this spice was being threatened by Guatemala. Indian productivity stood at 60 kg/ha, considerably less than Guatemala's 250 kg/ha. DBT, the Ministry of Commerce, the Spices Board and A V Thomas and Co in Kochi teamed up to field-test tissue-cultured cardamom. By 1991-92, elite tissue-cultured plantlets supplied by A V Thomas and Co and Indo-American Hybrid Seeds, Bangalore, had been planted and early results indicate tissue-cultured plants are increasing yield by about 65 per cent more than open-pollinated seedlings. The highest tissue-culture yield was 820 kg/ha, as compared to 312 kg/ha in open-pollinated plots.

A major DBT project was begun in 1989-90 for production of tissue-cultured tree species to provide firewood, fodder and timber. A DBT task force on production of biomass using tissue culture reported: "If the wood yield remains at its present figure of 0.7 tonnes per ha per year, the demand for fuel alone will lead to rapid deforestation. The provision of tree and shrub cover for degraded lands is one of the most urgent problems that the country is facing today. If seeds of uncertain genetic quality are used for planting -- as is often done -- the land will be held up for long periods under inferior, low-yielding plantations."

The report dismissed the argument that clonal forestry "will lead to annihilation in the event of a disease outbreak. The required genetic diversity can be introduced in clonal forestry by using highly productive but unrelated clones."

Following the report of the task force, DBT set up two pilot plants for large-scale production of tissue-cultured eucalyptus, teak, sandalwood, and narbans (Dendrocalamus strictus) at NCL, and various bamboo species, poplar, babul and rosewood at TERI. Other research institutes were assigned projects to develop tissue culture techniques for sal, sandalwood, poplar, babul, rosewood, alder, mahua, khejri and tiger bamboo (Bambusa vulgaris). But there have been delays in the setting up of pilot plants. The NCL plant, for example, became operational only in late 1992, two years later.

Since these projects were launched, 15,000 elite eucalyptus, teak and narbans plants have been transferred by NCL for field trials. Early results show tissue-cultured plants have a higher number of culms (stems) per clump and are taller than natural plants. About 80 per cent of the tissue-cultured plants became established. TERI scientists report good results with babul, khejri, jamun and arjuna (Terminalia arjuna).

The Ministry of Environment and Forests and state forest departments have identified seven states for conducting field trials, which are being monitored by two inter-state forest committees. However, full results of all these efforts will not be available for some time. Says NCL's Mascarenhas, "The utility of tissue culture will be determined only when the trees have grown to a specific height and survived well in the fields. For this, another 8-10 years will be required, as trees grow slowly."

But not everyone is happy with DBT's approach. Deepak Pental, dean of biological sciences at TERI, maintains besides being an unnecessarily expensive method, tissue culture for social forestry will be useful only when "legal issues like entitlement of wastelands to the poor are sorted out." Recalling TERI's experience with plantation on panchayat land in a village near Delhi, he said after TERI had taken pains to afforest it, the moment its lease lapsed, the trees were felled by the villagers. The forest department, Pental noted caustically, "is only interested in selling wood". Another complicating factor, says Pental, is that "nowhere in India is there a good germplasm collection for trees."

Pental, however, advocates tissue culture for floriculture or horticulture. "Floriculture technology is well developed, expert personnel is available and it is export-oriented." Horticulture, on the other hand, can dramatically transform India's nutritional situation. If elite material can be developed and given to farmers, we can get the next revolution in agriculture," says Pental.

DBT has also been moving towards horticultural and plantation crops. It has sanctioned a tissue culture project on mango and is considering projects on banana, citrus trees, gladiolus, chrysanthemum, carnation and rose. Projects have been taken up on coffee, cashew, pepper, nutmeg, cinnamon, clove, ginger, saffron, rubber, tea and cocoa. Tissue culture studies have also been initiated for regeneration of endangered mangrove species.

Among food crops, DBT-promoted projects involve four crops -- rice, turnips, chickpea and wheat -- and set up centres for plant molecular biology for this specific research at JNU; Madurai Kamaraj University; TNAU; National Botanical Research Institute, Lucknow; Osmania University and Bose Institute, Hyderabad.

NCL and JNU are working on chickpea and pigeonpea, both of which are in demand but have a low yield. NCL is experimenting with fusing wild varieties with cultivated ones and then using tissue culture techniques for regeneration. Raina is using tissue culture techniques to identify basmati rice varieties that can be grown easily in different conditions. Because basmati rice is not an easy crop to grow, some tissue-cultured varieties displaying basmati's characteristics of long kernel, fluffy grain and rich aroma are currently under IARI field trials.

On the whole, the tissue culture industry expects the response from foreign buyers to be better than from Indian farmers because of the latter's unfamiliarity with the new technology and its high cost. Nevertheless, the potential is enormous. Sivarama Reddy pointed out the promise of yields increasing by 30-40 per cent should compensate for the higher cost of the tissue-cultured plants.

Indian farmers can also be involved in a big way in orchid floriculture. Strawberry planters in Maharashtra have bought plantlets produced by Unicorn Biotek from elite Californian varieties. Tissue-cultured plants are virus-free though in the field, they tend to accumulate viruses that can reduce yield over time. This drawback is exploited by entrepreneurs because the farmer has to come back to them repeatedly for plant material. Not all tissue-cultured plants are expensive. Tissue-cultured pomegranate seedlings, for example, cost Rs 12 each whereas conventional seedlings cost Rs 15 each because pomegranate cuttings are hard to get.

NCL's Rajni Nadgauda insists if tissue culture is to benefit farmers, the government will have to play a key role and organise live demonstrations on a large scale to familiarise farmers with the new technology. The private sector, interested as it is in profits, cannot be expected to act as an intermediary.

K V Krishnamurthy, head of the plant tissue culture division of NCL, is confident tissue culture and genetic engineering can together transform Indian agriculture within a decade. But, he conceded, tissue culture cannot answer all problems for "only progressive farmers will invest in the costlier tissue-cultured plants, attracted by higher returns. Small farmers can only benefit if there are government subsidies". And, he noted, until such time, tissue culture techniques are likely to remain restricted to high-value plants like ornamentals.

But Kamaljit Singh argues the private sector can promote tissue culture among farmers by providing them with tissue-cultured plants and guaranteeing to buy back the products. "We could even have a cooperative scheme for farmers," Singh said.

Clearly, tissue culture technology is all set to take off. As the technology spreads from orchids and ornamentals there will be more employment and more foreign exchange earnings. But, there will also be a lot of heated thinking about industry and research labs working in tandem.

Reported by Anjani Khanna in Hyderabad, Shubhangi Mehrunkar in Pune and Delhi and S Gopikrishna Warrier in Madras.

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