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Biotechnology

Summing up qualities

Esalq develops an unprecedented technique for the production of plant hybrids beginning with species of passion fruit (maracujá) or of citrus fruit that are more resistant to illnesses

Four new varieties of passion flowers, obtained via a technique of genetic manipulation unheard of in Brazil, are being grown on the campus of the Luiz de Queiroz College of Agriculture (Esalq) of the University of São Paulo (USP), at the town of Piracicaba. They are plants originating from a process called somatic hybridization, which makes possible the fusion of the cells of cultivated species with wild species that are resistant to various diseases. Very different from the interspecific hybrids used in agriculture, which are obtained by crossings, these have the sum of the number of chromosomes of the species used in the cellular fusion. The technique has also been used to improve citrus fruit and promises good results for the control of diseases that frequently attack Brazilian orchards.

The new varieties could be used as stock in the growing of passion fruit (maracujá) and of other citrus fruit. Different from GMOs, obtained through the transformation of the cell with the introduction of genes originating from other species, the somatic hybrids are produced through the complete fusion of two isolated somatic cells of species of the same genre. Although, they have a more restrictive application – the technique does not bring with it suspicions of environmental risks or of health problems for man -, the somatic hybridization results in varieties that conserve all of the genetic characteristics of the two plants that originate it. The technique appeared in the 80’s as a method for the improvement of plants, and has been successfully implemented in citrus farming in the United States, France, Spain and Israel.

In Brazil, the first studies to improve the growing of passion fruit began some ten years ago under the coordination of professor Maria Lúcia Carneiro Vieira, of the Genetics Department of Esalq. Since then, the researcher has been dedicating herself to obtaining passion fruit graft that is more resistant, reducing the incidence of illnesses caused mainly by fungi. “If we consider the illnesses provoked by virus, bacteria and fungi together, they make up a loss of 35% in production of the close to 45,000 hectares cultivated throughout the country”, says Maria Lúcia. The projects, financed through FAPESP, already add up to investments of US$ 200,000.

The research for the improvement in citrus fruit is also making advances. Over the last five years, professor Francisco de Assis Alves Mourão Filho, of the Department of Vegetal Production, and professor Beatriz Januzzi Mendes, of the Vegetal Biotechnology Laboratory at the Nuclear Energy Center of Agriculture (Cena), have already produced eleven combinations of somatic hybrids with the objective of controlling illnesses in oranges. The research was developed through FAPESP’s Program of Young Research Support and has received investments of R$ 150,000.

Fruit juice industry
The passion flower is a plant native to South America. Of the 600 known species, close to 400 have their origins in various regions of Brazil. Only five of them are cultivated, those being the Passiflora edulis f. flavicarpa, the bitter-passion fruit, the main species used by farmers that is grown for the fruit juice industry and that makes the country the largest worldwide producer of passion fruit. “The juice of the passion fruit is the second most exported by Brazil – only losing out to orange juice -, and the main buying market is Europe where passion fruit is well appreciated for its taste considered to be exotic”, informs Maria Lúcia.

In spite of being very profitable – close to R$ 8,000 per hectare planted -, the growing of passion fruit has been facing serious problems due to the lack of selected genetic material, since the improvement programs are still very incipient. “We are dealing with a very recent domestication”, explains the researcher. For example, different from grape whose domestication stretches back thousands of years, passion fruit began to be cultivated after colonization, some five hundred years ago. With the introduction of monoculture, the species became to the target for many illnesses that considerable affect its production.The attack by illness over the last few years, provoked a rotation of the growth, which then acquired a nomadic character.

This characteristic also confronted another type of loss. The passion flower is planted on stakes in such a manner that the plant grows vertically and spreads to the sides, like a trellised grapevine. This implies a high cost in the implementation and management of the growth. “When a severe illness hits, the grower can lose all of his investment”, points out remembered Maria Lúcia. To avoid the lose of the plants during the rainy season, when the incidence of illnesses increases due to the high humidity and temperatures, the growers substitute all of the plants shortly after the first crop, planting everything all over again for the following crop, making the culture annual. “Though it doesn’t solve the problem, this is less onerous than the migratory character.”

The hope of the researcher for an efficient solution lies in the wild germoplasm. There are various collections of passion flowers in Brazil, for example, at the Embrapa Cassava and Fruit Company in the town of Cruz das Almas in the state of Bahia, and in the unit of Jaboticabal of the São Paulo State University (Unesp), with which Maria Lúcia maintains cooperation liaisons. “In these species resistance genes have already been identified with good potential for improving the performance of the commercial species”, she says.

The introduction of these genes could be done in various forms. One of them is through a crossing of the wild species with a cultivated one in order to obtain a sexual hybrid. “This had already been done a few years ago, but the majority of these hybrids were of no use as providers of pollen. In order to recover the productivity of the cultivated species, it would be necessary to have various back-crossings with the commercial species, which is impractical because the sexual hybrids are sterile”, explains Maria Lúcia.

Fusion pathways
The solution for obtaining plants that are more resistant came through the biotechnology of the system of hybridization. In the case of passion fruit, the cells are extracted from the leaves, both from the cultivated and the wild species. The success of the techniques depends on efficient mechanisms that promote cellular fusion, which can be done either through a chemical agent or shocks with an electric current. In both cases, the objective is to bring about the destruction of the cellular walls, which function as barriers blocking the fusion. The cells without walls, called protoplasts, are kept under controlled osmotic pressure in order to avoid the breakdown of the plasmatic membrane that would provoke the destruction of the cell.

After various processes of washing and centrifuging, the protoplasts are maintained in a culture medium, where the cellular wall is regenerated and the hybrid cells begin to multiply. Small colonies can originate from the one cell, which can be observed under the optical microscope. Normally, after 28 days the colonies can be seen by the naked eye and are transferred to a solid medium. They form themselves into an indistinguishable mass of cells, called callus, which can give rise to many seedlings. Each one of them will develop as a new plant, which still needs to pass through various analyses until it is guaranteed that one is dealing with a true somatic hybrid.

After having been selected, the somatic hybrids are cultivated until their adult phase, when they flower and bear fruit. The new plant keeps all of the genetic characteristics of the two original plants (the commercial and the wild). “This happens because the fusion creates a tetraploid genome, or that is to say, one which has double the number of chromosomes of the natural species”, explains Maria Lúcia.

Resistance to diseases
The first somatic hybrids were obtained in 1995 through the fusion of cells of the bitter-passion fruit (Passiflora edulis f. flavicarpa), which is the variety most cultivated, with cells from the wild species P. cincinnata, which is resistant to the bacteriosis caused by Xanthomonas sp. pv. passiflorae), P. giberti and P. alata, resistant to premature death, of etiology as yet unknown, which causes the deterioration of the plants. The species P. alata (or sweet-passion fruit) also shows resistance to wilt, a disease of the soil provoked by the fungus Fusarium oxysporum. “These hybrids tend to be more vigorous and to have leaves and stalks that are thicker, because their cells are larger”, explains the researcher.

“Nevertheless, they are not productive and their most important application is as a graft for a commercial variety.” Besides more vigorous, the expectation is that the somatic hybrid will have greater resistance to soil diseases and that it will present some mitigation of the symptoms provoked by the bacterium Xanthomonas, serving as protection for the crown used in grafting. The commercial passion fruit cuttings grafted onto the hybrids are showing good development both regarding the crown and as graft. The next step is to evaluate their performance as to productivity and resistance to diseases.

Gain in fruit growing
The expectation of the researchers is that the somatic hybridization promotes a very positive jump as well in the improvement of citrus fruit. In the opinion of professor Mourão Filho, conventional programs of improvement have been in existence for more than one hundred years, but the results are not good, because the citrus plants have some characteristics that make their improvement difficult. One of the problems is the high variability of the results of crossings. “When one carries out the pollination of a plant with a variety of another species, the off-spring vary considerably in their characteristics”, he stated.

“You can even have a plant with tolerance to drought or to certain diseases, but it loses these desirable characteristics of the original plant.” Another problem: due to a characteristic of the seeds of citrus plants, the germination gives origin not only to a zygotic embryo, which is the result of the pollen grain with the ovule, but also of various others that are identical to the mother. “It is as if you were going back to square one on improvement, because, at the moment of seed dispersion, plants identical to the mother reclaim themselves”, he affirmed.

The strategy adopted by the researchers was to obtain more resistant varieties to be used as stock in the field, the variety mostly in use today is the spiky-lemon (limão-cravo). Though it is very tolerant to drought and vigorous, it is very sensitive to deterioration, a disease of unknown cause that leads to the death of the plant, and to gummosis, a disease provoked by fungi. “However, there exist other stock seeds, such sweet-orange (laranja-doce) or country-orange (laranja-caipira), which are more tolerant to deterioration, though they are sensitive to drought. In this manner, our objective was to bring together genetic variations with complementary characteristics so as to improve the performance of the stocks”, explained professor Mourão Filho.

Currently, the eleven hybrids of citrus obtained through the project are in the phase of growth development. The next stage, which is being financed by the Paulista Fund for the Defense of Citrus Growing (Fundecitrus), is to make the stock and to take the plants into the field for an evaluation of their resistance to diseases and to obtain an index of productivity. The researchers are optimistic. “With absolute certainty, we are going to obtain good results for the citrus grower.”, affirmed professor Mourão Filho. However, the most important result has already been obtained: the domination of a technique that promises to bear more fruit over the coming years.

Transgenic pathway
As well as the somatic hybrids, professor Maria Lúcia is searching for alternatives for the improvement of passion fruit. One of them is the production of transgenic varieties, more tolerant to the bacteriosis brought on by Xanthomonas. The gene bactericida atacina A was extracted from Trichoplusia ni, a moth that frequently attacks cabbage, cauliflower and soy plantations. The transformation of the plant tissue was obtained through the bombarding with tungsten particles. Dozens of transgenic buds are in the phase of growing by way of a culture. According to professor Maria Lúcia, the research should take around five years in order to bring conclusive results, considering all of the procedures of bio security involved in the process.

Another battlefront against the Xanthomonas is the search for resistant genes in the exotic populations (outside of South America) of passion fruit. “We are carrying out a very promising crossing between a country variety, coming from Morocco, in Africa, and resistant to Xanthomonas, with a commercial variety (Maguary), susceptible to the bacteria”, related the researcher. The ninety plants obtained from this crossing were analyzed as to their DNA profile and a map of the linkage containing the markers existing on the DNA was constructed. This molecular map is the first in the world considering that one is dealing with a tropical fruit species. Also, for each individual plant its level of resistance to Xanthomonas was analyzed.

“We observed that there are individuals within the offspring population that demonstrate more resistance than the Moroccan father, indicating that there is the possibility of resistance to bacteriosis”, she affirmed. Now the ninety small plants are being transferred to a test field where they will be evaluated for their level of productivity. They make up part of a plant population of passion fruit that is going to give a new impulse to the growing of this fruit in Brazil.

The projects
1. Somatic Hybridization in Passion Fruit Flowers, Basic Studies and their Application in Genetic Improvement (nº 95/03029-0); Modality Regular Line of Research Assistance; Coordinator Maria Lúcia Carneiro Vieira – Esalq/ USP; Investment R$ 87,227.00
2. Production of Somatic Hybrids of Citrus Fruit through the Fusion of Protoplasts (nº 95/09044-0); Modality Regular Line of Research Assistance; Coordinator Francisco de Assis Alves Mourão Filho – Esalq/USP; Investment R$ 497,090.58

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