PART II: ADVANCES

CHAPTER 7
Exploiting the
Genetic Resources
of Rice in Argentina
through Population
Improvement

María Antonia Marassi^[9]
Juan Eduardo Marassi^[10]
Marc Châtel^[11]
Yolima Ospina^[12]

María Antonia Marassi

Abstract

Participatory plant breeding (PPB) is a new approach in genetic improvement. In contrast to conventional breeding, it aims to fully integrate farmers and other stakeholders of the production chain into the whole process of variety development. It also aims to decentralize, towards farm fields, the most important steps of selection and evaluation. PPB intends mainly to answer the needs of small farmers living in poor and marginal areas for whom conventional breeding cannot offer suitable varieties. Within the framework of a new research project, CIAT and CIRAD began, in April 2002, an upland rice PPB programme, using population improvement methods. Although currently implemented in Nicaragua and Honduras, plans are to extend the programme to other Central American and Caribbean countries. This chapter presents the strategies proposed for this work, emphasizing initial diagnosis and participatory varietal selection of available materials. The paper also describes stages of population improvement and extraction of improved lines to respond to the specific demands of each production zone. Finally, the expectations for the use of this methodology in the region are indicated.

Resumen

El fitomejoramiento participativo (FMP) es un nuevo enfoque del mejoramiento genético. En contraste con el mejoramiento convencional, busca integrar completamente a los productores y los otros grupos de interesados de la cadena de producción en todo el proceso de desarrollo de variedades. El también busca descentralizar hacia los campos de los agricultores los más importantes pasos de la selección y de la evaluación. Este enfoque también busca descentralizar, hacia los campos de los agricultores, los pasos más importantes de selección y evaluación. El FMP pretende principalmente responder a las necesidades de los pequeños campesinos de las zonas pobres y marginales a quienes el mejoramiento convencional no pudo ofrecer variedades. Dentro del marco de un nuevo proyecto de investigación entre el CIAT y el CIRAD, se propone desarrollar un programa de MP del arroz de secano para América Central y el Caribe con los métodos del mejoramiento poblacional. Este proyecto se inició en abril del 2002 en Nicaragua y Honduras. El objetivo de este capítulo es presentar las estrategias propuestas en ese trabajo enatizando las etapas iniciales de diagnóstico y de selección participativa con materiales ya disponibles; y presentar las etapas de mejoramiento poblacional y de extracción de líneas mejoradas para responder a las demandas específicas de cada zona de producción. Al final se incluyen las expectativas en cuanto a la utilización de esta metodología en la región.

Introduction

Exploiting the genetic resources of rice in Argentina through conventional techniques of genetic improvement has contributed significantly to increased crop production. These efforts have resulted in the development of innumerable varieties for the irrigated ecosystem that predominates in the country. The principal method of improvement used to develop these varieties was that of pedigree. Recently, several authors have suggested that this alternative seems to be taking breeding programmes towards diminishing genetic advances (Soares, 1992; Breseghello et al., 1999; Rangel et al., 2000) and, moreover, has helped narrow the genetic base of varieties released in Latin America (Cuevas- Pérez et al., 1992). Morais (1995) describes the sequence of progress of generations and the selection process by conventional improvement methods and highlights the results of these on genetic variability.

The implications of narrowing the genetic base, particularly the restricted use of genetic resources, are well known. Uniformity or lack of genetic variability in a commercial crop can lead to increased biotic and abiotic problems that would be difficult to solve.

According to Rangel and Neves (1997), one alternative for counteracting these limitations is to manage the genetic resources of rice by exploiting the germplasm, developing populations of broad genetic bases and using a breeding methodology that continuously accumulates favourable genes. Of the available methods that best fulfil these two aspects is population improvement through recurrent selection.

Likewise, the exchange of genetic resources between countries, including between a country and international research centres, is increasingly difficult. The introduction, development, and management of populations of broad genetic bases is another alternative - sometimes the only one - by which national programmes of genetic improvement can access new genetic diversity.

In Argentina, the exploitation of genetic resources through population improvement began in 1996, with the introduction and evaluation of three populations managed by the CIRAD/CIAT Rice Project in Cali, Colombia (Marassi et al., 2000). Based on this work, local populations were developed, introducing new genetic variability to the populations that performed best under the country’s conditions. These formed the basis on which the following activities (and whose progress is reported in this chapter) were carried out:

• Launching a population improvement programme, using the recurrent selection method

• Developing local populations and introducing new genetic variability to one of the initial populations

• Characterizing this new population

• Evaluating and selecting plants and the families obtained from these, and managing segregants originating from the populations being improved to produce improved lines and varieties for commercial distribution.

Developing and managing new populations

Introduced populations

In 1996, three populations were introduced into the country: PCT-6, PCT-7 and PCT-8. Taking into account their agronomic traits and performance under local cropping conditions during the 1996/97 cropping season, PCT-8 was selected as being the most adapted according to the data presented by Marassi et al. (2000). This population was subjected to a process of population improvement based on the evaluation and recombination of S_0:2 families.

To take advantage of the genetic variability and the male-sterility gene present in PCT-6 and PCT-7, two new populations were created by combining plants obtained only from these two base populations. Of the new populations, one (PARG-1) had a narrow genetic base and the other (PARG-2) a broad genetic base (Marassi et al., 2000). Both were developed with the principal purpose of generating fertile S₀ plants and permitting the development of fixed lines and production of commercial varieties. In the 1998/99 cropping season, 30 and 115 lines, respectively, were selected from these two new populations.

Developing populations of narrow genetic bases

To develop a population with a narrow genetic base (i.e. PARG-1\0\0\0), two populations, identified as having the least potential for the programme, that is, PCT-6 and PCT-7, were used, together with some lines selected from PCT-8. The development of this population was based on mixing the seeds of male-sterile plants that were harvested from the best-performing fertile S_0:1 lines, derived from fertile S₀ plants of each population. This work was carried out in Villaguay during the 1997/98 cropping season (Marassi et al., 2000). The concept of "a narrow genetic base" was assumed because the seed, mixed in equal proportions, were from only 5, 3 and 15 lines of populations PCT-6, PCT-7 and PCT-8, respectively. As observed, the necessary considerations of ideal sampling size to represent a population (Geraldi and Souza, 2000) were not taken into account in this case.

During 1998/99, the first recombination of the population was carried out by harvesting seed from a representative sample of the male-sterile plants. It should be emphasized that the idea was only to take advantage of the new variability for conventional improvement to develop commercial varieties. The new population was called PARG-1\0\0\1. In the 1999/00 and 2000/01 cropping seasons, similar work was carried out to obtain populations PARG-1/0/0/2 and PARG-1/0/0/3, respectively.

Developing populations of broad genetic bases

To develop a population with a broad genetic base (i.e. PARG-2\0\0\0), seeds were obtained from 80 selected male-sterile plants of the best-performing progeny of fertile S₀ plants of the three original populations (25 from PCT-6, 18 from PCT-7 and 37 from PCT-8) and mixed in equal proportions with seeds from the second recombination cycle of population PCT-8, identified as PCT-8\0\0\2 (Marassi et al., 2000). The mixture was prepared with equal quantities of seeds from the two groups, that is, from:

(1) PCT-6, PCT-7 and PCT-8
(2) PCT-8\0\0\2.

During 1999/00, the mixture was planted in the field and only the male-sterile plants were harvested, which made it possible to obtain the first recombination cycle, known as PARG-2\0\0\1. Likewise, during the 2000/01 cropping season, a second recombination cycle was obtained (PARG-2\0\0\2).

As already mentioned, this population was developed only to extract fertile plants and obtain fixed lines. For this same reason, this material became used only to select the best-performing fertile plants and for obtaining families by pedigree or anther culture.

Population PARG-3\0\0\0

The advances obtained in this stage, described by Marassi et al. (2000), made possible the production of population PARG-3\0\0\0. In the 1999/00 cropping season, seeds of this population were planted at CIAT, Colombia, where the first recombination (PARG-3\0\0\1) was carried out, the derived seeds of which were sent to Argentina. However, because this material arrived in the country only in March 2001, the first planting was carried out at Corrientes, in the greenhouse at the Universidad Nacional del Nordeste (UNNE), to conduct a preliminary characterization of the germplasm and a second recombination cycle, and to harvest plants for anther culture.

The process of population improvement through recurrent selection, based on the evaluation, selection and recombination of S_0:2 families and lines for anther culture, was initiated in the 2001/02 cropping season at Corrientes.

Characterizing population PARG-3\0\0\1

Even though it was outside the normal planting time for rice in the country, we decided to continue with the work of determining the changes that occurred in PCT-8\0\0\2 when new variability was introduced into it. For this, population PARG-3\0\0\1 was evaluated for phenotypic traits that were considered fundamental by the country’s breeding programmes. The hypothesis was that the changes had helped improve weaknesses observed in PCT-8\0\0\2, such as low vigour and excess plant height.

Population PARG-3\0\0\1 was planted, together with PCT-8\0\0\2 (its base population), at UNNE, Corrientes, during March 2001. Planting was manual, in 5-m-long rows, spaced at 0.15 m. Once plant height reached 0.20 m, irrigation was initiated, maintaining a water layer of 0.10 m until grain maturity. Neither fertilizers nor herbicides were applied; weed control was manual, and the low incidence of insect pests did not justify applying insecticides. The harvest of fertile plants in this population, and of those selected to be stabilized by anther culture, was carried out during August and September 2001.

Evaluations were carried out for vigour, days to 50% flowering, plant height and type, angle of flag leaf, panicle length, and grain sterility and type (length-width ratio), all based on scales developed by IRRI (1988) (Table 1).

It should be emphasized that both populations (PARG-3\0\0\1 and PCT-8\0\0\2), for which the traits are described below, were planted in the greenhouse. The evaluation for vigour was carried out according to a scale of 1 to 9, where 1 indicates excellent and 9 very poor germination. Population PARG-3\0\0\1 performed much better than PCT-8, as it presented a score of 3 for vigour versus the latter’s score of 7. Days to 50% flowering were 90, a period that is adequate for our conditions, although the population also presented notable extremes (70 to 125 days). Plant height ranged between semi-dwarf and normal for Argentine conditions, with an average population height of 95 cm, which is adaptable to the demands of improvement plans.

The angle between the tillers and the vertical axis was evaluated on a scale of 1 to 5, where 1 (open) refers to tillers presenting an angle of more than 70º against the vertical, and 5 (closed) to tillers presenting an angle of less than 45º against the vertical. Most of the population’s plants scored 3.5.

The IRRI scale (1988) was also used for the angle of the flag leaf, where 1 refers to an erect leaf and 7 to a falling leaf. Generally, the population scored 3.6, meaning that improvement would be necessary, as scores closer to 1 are preferred. Most flag leaves were also wide (>3 cm), a trait that was already observed in PCT-8\0\0\2.

Panicle length averaged 29.8 cm for the population. Panicle exsertion was evaluated on a scale of 1 to 7, where 1 indicated that the panicle base is well separated from the collar of the flag leaf and 7 the panicle base remains within the sheath of the flag leaf. The population scored an average of 4.3, that is, the panicles had not exserted much, or the panicle base was still within the flag leaf ’s collar. This trait had already been observed for PCT-8\0\0\2 and still requires being improved.

The degree of sterility was determined with a scale of 1 to 9, where 1 represented highly fertile panicles (more than 90%) and 9 completely sterile. The population scored 3.7, that is, more than 70% of spikelets in the panicles were fertile.

Table 1. Average, maximum and minimum values for the agronomic traits evaluated in rice populations PARG-3\0\0\1 and PCT-8\0\0\2 during 2001, UNNE, Corrientes, Argentina.

a. Standard deviation.
b. Data according to IRRI’s evaluation scale (1988).
c. Information collected from huskless grains

Figure 1. Vigour compared in rice populations PARG-3\0\0\1 and PCT-8\0\0\2 (base population of the first) on a scale of 1 to 9, where 1 indicates a vigorous plant and 9 a plant with poor vigour.

Shattering was evaluated on a scale of 1 to 9, where 1 indicates that shattering is less than 1% before maturity and 9 more than 50%. The population scored 4.5 (i.e. shattering before maturity was less than 6%), which represents progress over population PCT-8, which usually scored between 7 and 9, that is, shattering was more than 26% of grains in the panicle.

A trait of major importance to Argentine breeding programmes is grain type. The grains of the new population are predominantly long and slender, with a length-width ratio of more than 3, but less than 3.6. This implies an extremely long grain that would be discarded as entailing potential milling problems.

On analysing some of the scores obtained for population PARG-3\0\0\1 (Table 1), marked progress with respect to PCT-8 can be seen. Figure 1 shows that vigour, a negative trait in PCT-8\0\0\2, presented in PARG-3\0\0\1 a distribution where most plants concentrated in the classes of most interest to plant breeders.

For plant height (Figure 2) and days to 50% flowering (Figure 3), an improved distribution of plants is also observed, that is, more plants are found in the classes of most interest for improvement for irrigated conditions in Argentina. Generally, progress in terms of population PCT-8\0\0\2 can be observed, which progress will be accentuated even more, once the process of population improvement is initiated.

To compare preliminary studies conducted to characterize population PARG-3 by using molecular markers, Nei’s similarity index (1972) was used. The result of analysing 75 plants was an index of 0.40, whereas the commercial varieties currently planted in the country had an index of 0.60. This result indicates that the population has greater variability than does the group of commercial varieties. It is still necessary to compare analyses of the lines derived from the population to ensure that what was observed in the population is maintained at line level.

Figure 2. Comparing plant height for rice populations PARG-3\0\0\1 and PCT-8\0\0\2 (base population of the first).

Figure 3. Comparing days to 50% flowering for rice populations PARG-3\0\0\1 and PCT-8\0\0\2 (base population of the first).

Managing population PARG-3\0\0\1

Work on PARG-3\0\0\1 led to the extraction of 85 fertile S₀ plants that presented phenotypic traits appropriate for our breeding objectives. These materials were used in two ways: first, immature panicles were harvested to carry out in vitro anther culture to obtain homozygous lines. Of 40 S₀ plants that were selected for stabilization through anther culture, we could obtain, on the average, 25 plants, and derive at least 50 plants from each to produce a total of 1387 double-haploid plants. These lines were planted during the 2001/02 cropping season for seed production but 150 plants were lost, either for not having reached maturity due to poor development, or for their level of ploidy (haploids or triploids). Of the remaining 1237 plants, 757 plants were selected, mainly on the basis of their fertility, for use in a trial, together with fertile S₀ derivatives, to be conducted during the 2002/03 cropping season.

Table 2. Number of families (F) and plants (P) selected from rice populations PCT-6, PCT-7, PCT-8, PARG-1, PARG-2 and PARG-3 during the 1996/97 to 2001/02 cropping seasons in Villaguay, Entre Ríos, and from PARG-3 in the 2001/02 cropping season in Corrientes, Argentina.

a. Total number of selected S₀ plants, including those that were subjected to in vitro anther culture.

The second strategy used was to plant in rows the plants selected during the 2001/02 cropping season. We used 85 S₀ plants, some of which were in the group subjected to anther culture.

Both sets of lines - one originating from anther culture (757), and the other selected only for their agronomic traits (825) and derived from fertile S₀ plants selected in the 2000/01 cropping season - will be included in only one trial during the 2002/03 cropping season. The objective of this trial is to initiate population improvement for PARG-3\0\0\1.

Selection Strategies for Fixed Lines

S₀ plants derived from the original populations were used as described in

Table 2. PQUI-1 was introduced from Chile and was evaluated to assess its potential for our breeding programme. From each S₀ plant selected, S₁ panicles were planted in rows (S₁ family of panicles) to carry out phenotypic selection between and within families. From selected S₁, panicles from fertile plants were harvested for follow up by the traditional genealogical method and using the in vitro anther culture to accelerate the stabilization of selected materials.

The selection criteria used for S₀ plants of the different populations were days to 50% flowering (from 75 to 120), modern plant type, long slender or long wide grain type, panicles longer than 25 cm and good performance in tolerating diseases and iron toxicity.

Generally, the lines selected in this stage presented plant height of less than 1.20 m, 50% flowering between 80 and 110 days, flag leaf intermediate to erect, panicles longer than 28 cm and long slender grain type (length-width ratio between 3 and 3.8).

With respect to selecting materials with a long wide grain type from the populations, some materials are being kept because they present either glutinous characteristics (useful in certain markets) or an amylose content that is more than 22%.

Future plans

So far, Argentina has not completed recurrent cycles in the populations developed. All effort has concentrated on creating new populations and in characterizing population PARG-3\0\0\1.

During the 2001/02 cropping season, we evaluated the traits plant type, days to 50% flowering, grain type and quality and panicle length in a sample of more than 275 plants from population PARG-3\0\0\1.

The flow of materials in the coming years will be as follows: 2002/03 cropping season:

• Planting seeds derived from the 85 fertile plants selected from the population. The trial will also include seeds produced by double-haploid plants derived from the 25 selected plants and cultivated in vitro.

• Trial management and selection of the best lines by using Federer’s augmented block design with local checks, as proposed by Zimmermann (1997).

• Planting populations PARG-3\0\0\1, PCT-8\0\0\0 and PCT-8\0\0\2 to conduct studies:

  (1) with molecular markers (microsatellites) on the variability of these populations

  (2) to determine whether variations occur within them and in what sense once recombination and introduction of variability have been carried out.

• Planting advanced segregating lines derived from the populations, which will be managed through recurrent selection, together with advanced lines from the conventional improvement programme. All these lines will be evaluated together, using both morphology and microsatellite markers, to determine that variability contributing to population improvement in the national breeding programmes.

2003/04 cropping season:

• Evaluation of S_0:2 families to select materials for sending to CIAT for recombination and then returning to Argentina.

2004/05 cropping season:

• Evaluation of fertile plants selected from the best-performing families derived from the first recombination.

Given that only one generation per year can be carried out in the country, we will resort to CIAT, Colombia, to advance generations and carry out the corresponding recombinations.

Acknowledgements

We thank Laura Giarroco and Graciela Salerno for carrying out the variability analyses, using molecular markers. We furthermore thank the Corporación General de Alimentos S. A. and their field and laboratory staff for their collaboration in conducting, managing and evaluating the populations and their derivative lines.

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