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a Donald Danforth Plant Science Center, St. Louis, MO 63132
b Dep. of Biology, Washington University, St. Louis, MO 63130-4899
c CIAT, AA 6713, Cali, Colombia
d current address: United Soybean Board, St. Louis, MO 63141
* Corresponding author (p.chavarriaga{at}cgiar.org).
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONCENTERS OF ORIGIN AND...REPRODUCTIVE BIOLOGYCROSSESWEEDINESS AND INVASIVENESSREPRODUCTIVE ISOLATIONAREAS FOR FURTHER RESEARCHREFERENCES |
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Abbreviations: CIAT, Centro Internacional de Agricultura Tropical • GM, genetically modified • PBS, Program for Biosafety Systems • USAID, U.S. Agency for International Development
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONCENTERS OF ORIGIN AND...REPRODUCTIVE BIOLOGYCROSSESWEEDINESS AND INVASIVENESSREPRODUCTIVE ISOLATIONAREAS FOR FURTHER RESEARCHREFERENCES |
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Received for publication May 18, 2007.
a Donald Danforth Plant Science Center, St. Louis, MO 63132
b Dep. of Biology, Washington University, St. Louis, MO 63130-4899
c CIAT, AA 6713, Cali, Colombia
d current address: United Soybean Board, St. Louis, MO 63141
* Corresponding author (p.chavarriaga{at}cgiar.org).
Cassava (Manihot esculenta Crantz) is a vitally important food source for many people in developing tropical countries. There are significant opportunities for improving the compositional qualities and pest resistance of cassava, and modern biotechnology is expected to play an important role in these improvements. The testing and development of genetically modified cassava will of course be subject to regulatory review, and experimental field trials must be performed in a fashion that prevents gene flow from the regulated plants. Methods to ensure reproductive isolation will be derived from a fundamental understanding of the biology of the crop. A current and comprehensive document on cassava reproductive biology is not yet available but is essential to guide regulators and scientists in planning and evaluating measures for reproductive isolation of confined field trials. This paper compiles a current view of the reproductive biology of cassava for use in experimental design and regulation of confined field trials. With the current state of knowledge on gene flow and seed dormancy in cassava, three methods for reproductive isolation of regulated experimental plots may currently be recommended: (i) removal of flower buds before flowering, (ii) destruction of plants before flowering, and (iii) floral bagging to contain pollen and seed. Areas for further research in cassava biology and biosafety are suggested.
Abbreviations: CIAT, Centro Internacional de Agricultura Tropical • GM, genetically modified • PBS, Program for Biosafety Systems • USAID, U.S. Agency for International Development
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONCENTERS OF ORIGIN AND...REPRODUCTIVE BIOLOGYCROSSESWEEDINESS AND INVASIVENESSREPRODUCTIVE ISOLATIONAREAS FOR FURTHER RESEARCHREFERENCES |
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There are many significant opportunities for improving cassava, especially in terms of nutritional qualities, reduction of cyanogenic content, pest resistance, and compositional qualities (Taylor et al., 2004). Progress in these areas stands to greatly benefit the many subsistence farmers and their families who rely on cassava and its products (Kawano, 2003). Traditional breeding of cassava is constrained by a number of intrinsic factors, including high levels of genetic heterozygosity, variable flowering patterns, and low seed set and germination (Jennings and Iglesias, 2002). Because of these difficulties, modern biotechnology and especially genetic modification through recombinant DNA methodology may be expected to play a significant—indeed, an essential—role in future improvements of cassava.
In March 2004, a meeting of the Cassava Biotechnology Network, an international group of scientists involved in research on cassava biotechnology, took place at the Centro Internacional de Agricultura Tropical (CIAT), Colombia. The group discussed recent efforts to conduct field trials of genetically modified (GM) cassava plants in Africa and Latin America. Members identified issues of biosafety with regards to the design and execution of regulated and confined field trials as a key to facilitating future research on GM cassava. In particular, a compilation of current information on reproductive biology and reproductive isolation was requested to establish sound procedures to prevent gene flow from regulated field trials of GM cassava.
As a result of these discussions, CIAT requested assistance from the Regulatory Approval Strategies component of the Program for Biosafety Systems in developing a document to address this critical need. The Program for Biosafety Systems (PBS) is a biosafety consultancy funded by the U.S. Agency for International Development (USAID); the Regulatory Approval Strategies component of PBS is housed at the Donald Danforth Plant Science Center in St. Louis, MO. The Centro Internacional de Agricultura Tropical expressed the hope that this document would be "an essential resource for developing country institutions and their partners when assembling biosafety dossiers—ensuring soundness and a degree of consistency for those aspects of transgenic cassava testing that are not construct-specific" (L.T. Kent, personal communication, 2005). The present review of reproductive biology and suggested measures for reproductive isolation of regulated and confined field trials were undertaken by PBS in response to CIAT's request.
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CENTERS OF ORIGIN AND DIVERSITY |
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TOPNOTESABSTRACTINTRODUCTIONCENTERS OF ORIGIN AND...REPRODUCTIVE BIOLOGYCROSSESWEEDINESS AND INVASIVENESSREPRODUCTIVE ISOLATIONAREAS FOR FURTHER RESEARCHREFERENCES |
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The 98 known wild species of the New World genus Manihot are distributed across warm regions of the Americas, from southern Arizona to Argentina (Rogers and Appan, 1973). There are two centers of species diversity in the genus; most species occur in northern South America (
80 species), and a secondary center of diversity occurs in Mexico and Central America (17 species, plus the related taxon Manihotoides pauciflora). A list of Manihot and Manihotoides species and their approximate geographical distributions are shown in Table 1
. Central Brazil has the highest diversity of Manihot species and is home to about 40 wild species. Most Manihot species occur in dry or seasonally dry conditions. Although a few species are found in rainforests, they tend to be sporadic in their distributions and never become dominant members of the local vegetation (Rogers and Appan, 1973). The growth habit of Manihot species ranges from low herbaceous vines to trees exceeding 12 m in height (Rogers and Appan, 1973).
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REPRODUCTIVE BIOLOGY |
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TOPNOTESABSTRACTINTRODUCTIONCENTERS OF ORIGIN AND...REPRODUCTIVE BIOLOGYCROSSESWEEDINESS AND INVASIVENESSREPRODUCTIVE ISOLATIONAREAS FOR FURTHER RESEARCHREFERENCES |
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Flowering is also dependent on plant habit. A flower bud typically forms when the plant branches, so that more highly branched genotypes flower more prolifically than those with a sparsely branched habit. Since flower-bud formation is preceded by apical branching, a prominent visual indication of incipient flowering is available to identify plants in the immediate preflowering stage.
The following is a general scheme of the flowering process, as observed by experienced breeders at CIAT:
Pollen
The pollen grains of cassava are relatively large in size and are sticky. Wind pollination therefore appears to be of little consequence (Rogers and Appan, 1973), with several species of wasp (mainly Polistes spp.) and honeybees (Apis mellifera) considered the main pollinators in Colombia and Africa, respectively (Kawano, 1980). Cassava pollen shows size dimorphism within the same genotype, the larger grains being 130 to 150 µm in diameter, whereas the smaller grains range from 90 to 110 µm. In some clones, the larger grains are more abundant, whereas in other clones the smaller grains are more common. The larger pollen grains have been observed to have better in vitro germination (60% germination after 2 h at 40°C) than the smaller ones, which may have less than 20% germination (Plazas, 1991).
Cassava pollen loses viability rapidly after it is shed. Leyton (1993) found 97% seed set with pollen used immediately after its collection, 56% seed set with pollen stored for 24 h at 25°C, and 0.9% seed set (one seed from 102 pollinations) after 48 h of storage. In practice, breeders take care to perform pollinations within 1 h after collection of pollen to help ensure successful fertilization; pollen viability seems to decline substantially after this time (P. Chavarriaga and N. Morante, personal observation, 2005).
Seed Characteristics
Developing seeds are viable 2 mo after pollination, and the fruit becomes mature about 1 mo after that, or about 3 mo after pollination (Ceballos et al., 2002). The fruit is a trilocular schizocarp, and seeds are ovoid-ellipsoidal, approximately 100 mm long and 4 to 6 mm thick (Alves, 2002). Dehiscence is explosive; the seed initially falls close to the mother plant but then may be further dispersed by ants, which carry seeds to their underground nests. Through these two mechanisms of autochory followed by myrmecochory, a seed may be dispersed up to several meters from its place of origin (Elias and McKey, 2000; Elias et al., 2001).
Seed production and viability are variable, depending largely on the vigor and number of flowers borne by the parent plant (Kawano, 1980). Jennings (1963) reports that one viable seed per fruit is normally achieved in controlled pollinations, from a maximum of three possible in the trilocular ovary. Ceballos et al. (2004) indicate that one to two viable seeds are obtained from each hand pollination. Newly harvested seeds exhibit physiological dormancy and require 3 to 6 mo of storage at ambient temperature before they will germinate (Jennings and Iglesias, 2002).
Cassava seeds are adapted to ant dispersal, with large energy reserves that allow deep burial and a long dormancy period (Pujol et al., 2002). Seeds can remain viable when stored under ambient conditions for up to 1 yr, although germination percentages may decline substantially after 6 mo (Rajendran et al., 2000). Under cool-temperature storage conditions (4°C and 70–80% relative humidity) seeds have been known to survive for up to 7 yr with no loss of germination (N. Morante, personal communication, 2005). The persistence of natural seed banks has not been well documented, but they may endure for many years (Elias et al., 2000).
Seed germination is favored by dry heat and complete darkness. Ellis et al. (1982), working with two-dimensional temperature gradient plates, found that germination occurred most often when temperatures exceeded 30°C for part of the day, with a mean temperature of at least 24°C. They suggest that an alternating temperature regime of 30°C for 8 h and 38°C for 16 h for at least 21 d is the most appropriate for determining cassava seed viability under laboratory conditions.
The combination of deep burial by ants, a long dormancy period, and heat-activated germination suggests that the ancestor of cassava may have evolved under conditions of sporadic natural fire and was thus uniquely suited to domestication under slash and burn agriculture (Pujol et al., 2002). In this view, the seed bank of ancestral cassava, protected from environmental hazards by deep burial and physiological dormancy, could await the triggering effect of lightning-caused fires, allowing seedlings to avoid competition with established vegetation. In addition to seed, the rootstocks of the crop's ancestor were also likely well adapted to fire; the wild relative M. esculenta subsp. flabellifolia has been observed to show vigorous regrowth from rootstocks in areas of burned forest (K. Olsen, personal observation, 2005). Domesticated cassava was thus "pre-adapted to slash and burn agriculture, which enabled spread of this plant into habitats much wetter than those occupied by its wild ancestors. [Further,] cultivation by stem cuttings may have originated via attempts by foraging peoples to supplement the density in newly burned areas" (Pujol et al., 2002, p.377).
Botanical seed is not usually used for commercial propagation of cassava. Genetically, any particular cassava clone is highly heterozygous, and propagation from sexual seed results in wide and unpredictable diversity of phenotypes, which is of interest to breeders but presents difficulties in propagation (Ceballos et al., 2004). Propagation of cassava is therefore accomplished by vegetative stem cuttings to preserve the known characteristics of favored clones, as described below. Amerindian peoples of South America frequently encourage volunteer seedlings in their native gardens, in the hope of selecting a superior clone, which is then propagated vegetatively (Salick et al., 1997; Elias et al., 2000; Elias et al., 2001). Heterotic volunteer seedlings resulting from natural outcrosses are preferentially retained, since they are larger and much more vigorous than inbred seedlings, which can suffer from inbreeding depression (Kawano, 1980). This practice contributes to the maintenance of genetic diversity in cultivated populations (Pujol et al., 2005). Seedlings are initially smaller than plants developed from vegetative cuttings and require special care to become established and prosper.
Vegetative Propagation
Cassava is normally propagated by means of stem cuttings, which are known horticulturally as ‘stakes’. Stakes are typically at least 20 cm long, and have 4 to 5 nodes each with a viable bud. Stakes must be transported carefully to avoid damage and may be treated with agrochemicals to prevent pest or disease establishment in the new plants (Leihner, 2002).
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CROSSES |
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TOPNOTESABSTRACTINTRODUCTIONCENTERS OF ORIGIN AND...REPRODUCTIVE BIOLOGYCROSSESWEEDINESS AND INVASIVENESSREPRODUCTIVE ISOLATIONAREAS FOR FURTHER RESEARCHREFERENCES |
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The reproductive isolation of different stands of cultivated cassava is of interest in conventional breeding programs, to maintain the characteristics of different breeding lines, as well as in the confinement of trials of GM cassava. Limited information is available on natural gene flow and associated distances required for reproductive isolation in cassava.
Results from the empirical experience of cassava breeders have been used in the past to help define isolation requirements. While working at CIAT, Kawano et al. (1978) determined that a 30-m isolation distance was sufficient to avoid cross-pollination, and a distance of 30 m between stands of different clones became the established standard of breeders at CIAT to prevent cross-pollination (CIAT, 1974). Genetic isolation of test plots in conventional breeding programs at CIAT is also accomplished using an arrangement of 4 m of alley, 8 m of androsterile cassava, and another 4 m alley, for a total of 16 m distance (P. Chavarriaga and N. Morante, personal observation, 2005). Additionally, based on extensive personal experience in breeding programs, Kawano (1980) suggested that 500 m is an appropriate distance for "perfect isolation" of two populations in genetic studies. However, these standards were based on visual observations and were established before the advent of modern molecular techniques. New methods are now available to quantify more precisely the gene flow between stands of cultivated cassava and to elucidate the factors influencing it.
It is important to note that because of the typical vegetative propagation used for cassava, the consequences of gene flow from either experimental or commercial material are likely to be of less concern than with other crops. Even if a low level of successful outcrossing were to occur from experimental plants to the flowers of neighboring cassava, any novel traits would not be passed on in the typical vegetative propagation system used for cassava.
Interspecific Crosses: Wild Relatives
All Manihot species, including cultivated cassava, that have been studied so far have a chromosome number of 2n = 36 and show regular bivalent pairing at meiosis (Jennings and Iglesias, 2002). Thus, species karyotypes do not rule out the possibility of interspecific hybridization among Manihot species. However, such hybridization appears to be uncommon. Substantial work has been undertaken attempting to artificially introgress genes from wild species into cultivated cassava for breeding purposes (Nassar, 1989, 2003; Nassar et al., 1986; Hahn et al., 1990), and such efforts have met with mixed success. Nassar (2003), for example, reported no fruit set and no viable seed from 145 flowers of M. pohlii hand pollinated with pollen of cassava. Natural (insect-mediated) crosses of cassava with M. neusana and M anomala were more successful than hand pollinations but still resulted in less than 5% hybrid seed from an arrangement in which rows of cassava (as the pollen parent) were alternated with rows of the wild species (Nassar, 1989).
The more closely related the wild species is to cultivated cassava, the more successful hybridization seems to become—16 successful crosses at CIAT between cassava and the conspecific wild progenitor M. esculenta subsp. flabellifolia resulted in "thousands of seeds," whereas only five seeds of unknown viability were obtained from two crosses with M. aesculifolia, according to Roa et al. (1997, p. 748).
The natural hybridization of cassava with its closely related wild relatives has been reported and confirmed using modern molecular methods (Second et al., 1997). Natural hybrids with M. esculenta subsp. flabellifolia or M. pruinosa have been identified as arising from feral cassava populations surviving on the margin of an abandoned cassava plantation in French Guiana (Duputié, 2004; Léotard and McKey, 2004; Duputié et al., 2007). The sexually compatible wild relatives in this case are either the immediate ancestor of cultivated cassava or a closely related species. From the results of both artificial and natural hybridizations, it seems likely that genetic or physiological factors play a significant role in restricting gene flow from cassava to related populations. The probability of gene flow, as well as the stringency of measures required to prevent it, may thus diminish rapidly with increasing evolutionary distance between the species. Most Manihot species do not hybridize readily with cassava (Olsen and Schaal, 2001), and thus it cannot be assumed that such hybridization is common in nature. Nonetheless, the potential for interspecific introgression between cassava and wild Manihot species should not be discounted.
Manihot glaziovii (Ceara rubber tree) is the only relative of cassava that is reported to be naturalized in Africa. M. glaziovii is thought to be closely related to M. esculenta (Rogers and Appan, 1973; Second et al., 1997), and hybrids between cassava and M. glaziovii are highly fertile (Nassar, 1982). Natural hybrids between cassava and M. glaziovii, identified by morphological and electrophoretic markers (Wanyera et al., 1994) and DNA-based restriction fragment length polymorphism (RFLP) markers (Beeching et al., 1993), have been collected in Africa. Certain African cultivars can also be identified as descendents of M. glaziovii hybrids by the same technique (Beeching et al., 1993). Naturally occurring hybrid stands have been reported (Lefevre, 1988). These reports reflect hybridization presumably occurring with the two species in close proximity over long periods of time; the probability of gene flow from a particular stand of cassava to M. glaziovii over specific distances and a finite time period, as would be the case with an experimental confined field trial, remains to be established.
Manihot glaziovii seems to be widely distributed in other parts of the tropics as well. Rogers and Appan (1973) have reported collections in Asia from Laos, Sri Lanka, Malaysia, Indonesia, Philippines, and India, as well as from the New World tropics and islands in the Pacific Ocean.
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WEEDINESS AND INVASIVENESS |
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TOPNOTESABSTRACTINTRODUCTIONCENTERS OF ORIGIN AND...REPRODUCTIVE BIOLOGYCROSSESWEEDINESS AND INVASIVENESSREPRODUCTIVE ISOLATIONAREAS FOR FURTHER RESEARCHREFERENCES |
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The low fecundity and physiological dormancy of seeds also limits the spread and establishment of the crop into unmanaged habitats. Feral stands of cassava are reported to exist on the margins of abandoned plantations in South America, and other Manihot species are known to naturalize over time, such as M. glaziovii in Africa. However, cassava is not considered to be a weed in agricultural settings and is not invasive. Evaluated against Baker's (1965) 12 characteristics of weeds, only one, "discontinuous germination and long-lived seeds," appears to apply unreservedly to cassava.
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REPRODUCTIVE ISOLATION |
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TOPNOTESABSTRACTINTRODUCTIONCENTERS OF ORIGIN AND...REPRODUCTIVE BIOLOGYCROSSESWEEDINESS AND INVASIVENESSREPRODUCTIVE ISOLATIONAREAS FOR FURTHER RESEARCHREFERENCES |
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Each of the following sections provides a set of procedures to ensure reproductive isolation of cassava. A researcher would choose to follow the procedures described in one of the three options below, depending on the objectives of the trial:
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Control of regrowth from vegetative parts may be accomplished by herbicides, by burning, by burying, or by otherwise devitalizing the vegetative parts, such as chopping or grinding with commercial equipment designed for this purpose. If deep burial is used, it is recommended that vegetative material be covered with soil to a depth of at least 30 cm to prevent reemergence. Mature vegetation may be chopped before burial or burning, which should help destroy the material, speed decomposition, and prevent regrowth. Generally, the plot area should be monitored for regrowth for several months, or sufficient time to allow regrowth under local rainfall patterns. It is recommended that any crop to be grown in the area immediately after the regulated trial be chosen so as not to interfere with the recognition and destruction of regrowth of the experimental plants.
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AREAS FOR FURTHER RESEARCH |
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TOPNOTESABSTRACTINTRODUCTIONCENTERS OF ORIGIN AND...REPRODUCTIVE BIOLOGYCROSSESWEEDINESS AND INVASIVENESSREPRODUCTIVE ISOLATIONAREAS FOR FURTHER RESEARCHREFERENCES |
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Experimental evaluation of gene flow from cassava to specific wild relatives in nature, as related to the physical and genetic proximity of the plants, is needed both to design safe experimental trials and to guide future risk management decisions that will be required before any general release of GM cassava. The risk of gene flow under natural conditions may be limited to a specific subset of wild relatives or to specific conditions, due to the natural constraints discussed above. Until the true nature of the biosafety issues posed by the development of new and potentially GM cassava lines is known, the measures of reproductive isolation described here should be applied to experimental trials, especially those with regulated GM plants.
The authors are grateful to Professor Nagib Nassar (Universidade de Brasilia, Brazil) and Dr. Donald McKenzie (AgBios, Ottawa, Canada) for their work on an early version of this paper. The following colleagues gave helpful suggestions on the manuscript: Dr. Hernan Ceballos (CIAT, Cali, Colombia), Dr. Bruce Macbryde (USDA-APHIS, Washington, DC), Dr. Claude Fauquet (Donald Danforth Plant Science Center, St. Louis, MO, USA). K.M. Olsen assembled the data presented in Table 1 while serving as a paid consultant of the Donald Danforth Plant Science Center. These data were published previously in the document "Cassava (Manihot esculenta Crantz): Reproductive biology and practices for confinement of experimental field trials," by P. Chavarriaga-Aguirre and M. Halsey, report prepared for the Regulatory Approval Strategies of the Program for Biosafety Systems: Washington, DC. The Regulatory Approval Strategies Component is managed by the Donald Danforth Plant Science Center under subagreement EEM-A-00-03-00001-00-(DDPSC)-2826-30X with the International Food Policy Research Institute, which manages the overall PBS program.
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.
Received for publication May 18, 2007.
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TOPNOTESABSTRACTINTRODUCTIONCENTERS OF ORIGIN AND...REPRODUCTIVE BIOLOGYCROSSESWEEDINESS AND INVASIVENESSREPRODUCTIVE ISOLATIONAREAS FOR FURTHER RESEARCHREFERENCES |
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