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Plant Evolution: De-evolution and re-evolution of maize
ELLIOT M. MEYEROWITZ
PLANT EVOLUTION
De-evolution and re-evolution of maize Studies of the genetic differences between maize and its wild predecessor have started to reveal genes responsible for evolutionary innovations. Maize was derived by human selection from a wild progenitor called teosinte, a grass that grows in southern Mexico and Guatemala. Both plants are taxonomically termed Zea mays, though they are given different subspecific designations, with maize being Zea mays subsp. mays, and the strain of teosinte most closely related to maize designated Zea mays subsp. parviglumis [1]. Despite their close relationship, maize and teosinte have major structural, and therefore developmental, differences. Elucidation of the molecular basis of these differences could shed interesting light on the evolutionary origin of novel morphological features in plants. Studies with this aim in mind are beginning to bear fruit. Although maize and teosinte closely resemble each other in their stem, leaf and root structures and in their male inflorescences (tassels), there are several important differences between them (Fig. 1). Maize has a single primary stem with a tassel on top, whereas teosinte is branched, and has a tassel not only on the
top of the main stem, but also at the apex of lateral branches. The female inflorescence of maize is the familiar ear, consisting of a cob covered with kernels (fruits containing a single seed). The simplest maize ears are four-ranked, with two kernels per rank due to the development of paired spikelet primordia. They thus have a minimum of eight rows of kernels around the cob circumference. The female inflorescence of teosinte consists of a slender rachis (an ordinary stem, quite unlike a cob) with seeds projecting from it on only two sides. This inflorescence is two-ranked, with only one kernel per rank due to the abortion of one of the two spikelet primordia [2]. Maize kernels are naked on the cob, with only a small, papery chaff surrounding the base of each kernel. Teosinte kernels are covered on one side by outgrowths of rachis tissue, and on the other by a tough and extremely hard glume. They are consequently inaccessible and inedible except when very young, before the glume hardens, or when somehow extracted from
Fig. 1. Comparison of a modern maize plant, maize ear and a pair of maize kernels (left) with a teosinte plant, teosinte ear and teosinte kernels (right). (Adapted from [5].)
©Current Biology 1994, Vol 4 No 2
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Current Biology 1994, Vol 4 No 2 the glume. In addition, maize seeds stay on the plant when mature (a key characteristic of seed crops), whereas the kernels of teosinte, like those of any wild plant, disperse when they are mature. Maize cannot survive without human intervention because of its inability to disperse its seeds, and because of the vulnerability of the naked seeds to insects and rodents. Maize is thus a genetically engineered plant, which lives only as a human symbiont. Despite the remarkable morphological differences between maize and teosinte, we know that they are extremely close relatives. The archeological evidence indicates that native Americans derived maize from teosinte less than 10000 years ago. Both maize and teosinte have a haploid content of ten chromosomes, which have the same lengths and arm ratios, and heterochromatic knobs in the same relative positions 131. Analyses of isozymes and of chloroplast DNA restriction fragments have shown that maize and teosinte are genetically very similar - far more similar to each other than either is to other related plants [1]. The two plants are completely compatible in crosses,
with normal recombination occurring over most or all chromosome intervals [4]. This leaves us with the profound genetic question of how maize was created from teosinte. What sorts of mutations were obtained, and bred together, to create a new plant architecture, the novel structure of the ear and a naked fruit? That is, what is the nature of the genetic differences between the two close relatives that leads them to have such different patterns of development? This is a restatement of a central question in evolution - what is the molecular and genetic mechanism by which morphological innovation occurs? The genetic basis of the origin of maize was first successfully approached by Beadle, who crossed a primitive race of maize to teosinte and then self fertilized the F1 hybrid progeny. In a large population of the resulting F2 progeny, Beadle found that the parental phenotypes reappeared at a frequency of around 1/500, indicating that the differences between maize and teosinte might be due to as few as five major unlinked Mendelian loci [5]. Earlier (and subsequent) crosses between modern agricultural maize varieties and teosinte gave far more complex results, probably because there are at least as many genetic differences between primitive, small-eared varieties of maize and their enormous modern descendants as there are between teosinte and the primitive maize races [6]. Beadle's experiment could not, however, identify the genetic map locations of the genes that made teosinte into maize, because the genetic marker lines of maize have the background of modern varieties. In addition, several different genetic regions contribute to certain of the single morphological differences between teosinte and maize, making a Mendelian analysis of the differences difficult. The problems of inadequate genetic markers in primitive maize, and of multigenic inheritance, have now been solved and Beadle's analysis repeated. The problem of genetic markers for the mapping of differences in teosinte and maize has been solved by the use of restriction fragment length polymorphism (RFLP) markers. Teosinte and maize (and different maize varieties) show abundant RFLPs, which have been mapped across the entire genome. The problem of analyzing multigenic inheritance has been solved by application of new statistical methods for quantitative trait locus (QTL) mapping. Doebley and his co-workers [7,8] have used RFLPs and QTL mapping to define and genetically map the differences between teosinte and a primitive maize variety, and have found that, consistent with Beadle's result, five major QTLs control most of the morphological differences between teosinte and its descendant.
Fig. 2. Photograph of a teosinte rachis with its two rows of kernels (left) next to the rachis of a teosinte plant into which the maize Tgal allele has been introgressed (right). Note the reduction in the cupule and the outward growth of the glume caused by the maize gene. (Photograph courtesy of John Doebley.)
The difference between the single stem and single tassel of maize, and the branched architecture and multiple tassels of teosinte, is largely controlled by a small region on chromosome arm 1L, the maize copy of which dominantly, or co-dominantly, confers the maize
DISPATCH
branching pattern. There is a known recessive genetic mutation in modern maize that mimics the teosinte condition, teosinte branched (tbl), and this maps to the same chromosome arm as the branching pattern determinant. Most of the difference between the soft glumes of maize and the extremely hard glumes of teosinte correlates with a genomic region near the centromere on the short arm of chromosome 4. Again, the maize genetic region acts dominantly or co-dominantly to the homologous teosinte region. The difference between the two-ranked teosinte female inflorescence and the many-ranked maize ear depends on the paired floral spikelets of maize (teosinte also makes paired spikelet primordia, but one member of each pair aborts), as well as on there being more ranks of spikelet pairs in maize. The paired spikelets map predominantly to locations on chromosomes 1 and 3L, whereas the inflorescence phyllotaxy (rank) depends to a large degree on a portion of chromosome arm 2S, and a region on chromosome 5. The ease with which teosinte kernels fall from the plant depends on a region of chromosome arm 1L, with a contribution from other regions that may vary depending on the race of primitive maize used in the crosses (this character may thus have independently evolved from the teosinte condition in different maize lines). Five genomic regions are therefore responsible for the greatest part of the striking morphological differences between teosinte and maize. Do each of these regions represent single genes? What is the phenotypic effect of each of these regions alone - that is, what does teosinte look like with each of the single maize chromosomal loci, and conversely, what does maize look like with each of the single teosinte loci? The answer to these questions has now been obtained [9] for the chromosome 4 region that is largely responsible for the small and soft glumes of maize. Using RFLPs to speed the process, the teosinte version of this region has been introgressed into a maize genetic background, and vice versa. Analysis of the resulting near-isogenic lines has demonstrated that the chromosome 4S region contains a single gene affecting the glume. This gene has been named teosinte glume architecture 1 (tgal). The maize allele (Tgal) in a teosinte background makes the cupule - the indented part of the rachis that covers one side of the seed shallow, and makes the glume that normally covers the other side of the seed point outward at maturity, rather than curving over the seed (Fig. 2). The net effect is that the seed angles out from the rachis, and is partially exposed. In the converse strain, a de-evolved maize variety with the teosinte (tgal) allele in a maize background, the normally small and soft glume of maize is harder and thicker, and grows in a way that causes it to curve over, and partly cover, the kernel (Fig. 3). This work for the first time reveals the genetic nature and phenotypic effect of a single one of the small number of genes responsible for the evolution of maize from teosinte, and shows how the individual effects of the other
Fig. 3. Comparison of maize ears (without kernels). On the left is an ear homozygous for the maize Tgal allele; the ear on the right is homozygous for the teosinte tga I allele. The outer glumes of the ear on the left are thin and perpendicular to the ear axis, whereas those of the ear on the right are thick and curved upward. (Photograph courtesy of John Doebley.)
genes can also be elucidated. The molecular cloning of Tgal is now possible, and should soon reveal the nature of the gene product and of the mutation that distinguishes the teosinte and maize alleles. The Tgal results also shed light on an ethnobotanical mystery: why would people start to grow and breed something as useless as teosinte seems to be? As Kempton put it in 1937, "No more useless grasses from the standpoint of human consumption could be devised than the American relatives of maize" (quoted in [10]). We can consider two hypotheses. One is that there is a use for teosinte that would give a reason to cultivate the plant. Beadle [10] reported that he could pop the kernels of teosinte to make popcorn. The kernels might thus have been desirable enough to serve as encouragement for propagation, at least if the early Mexicans were, like Beadle, extraordinarily fond of popcorn (N. Horowitz, personal communication). Beadle [5] later reported experiments in which he ground and ate mature teosinte kernels and did not get sick. An alternative hypothesis is that the use of teosinte as food came only after one or more of the mutations that separate maize and teosinte were present in a local teosinte population. Tgal is an excellent candidate for such a gene: this allele alone, in a genetic background that is otherwise that of teosinte, markedly alters the glumes and, by changing the shape of cupules and glumes, exposes the seeds. Tgal mutant teosinte may thus have been the original crop, to which the other mutations were later added. One question raised by the recent genetic studies of teosinte is whether we can soon re-evolve maize. Could one find populations of teosinte that are segregating for
129
130
Current Biology 1994, Vol 4 No 2 the mutant alleles that have been fixed to create maize? Could one use RFLP markers to identify plants carrying the mutant alleles, which could be crossed to reconstitute maize from its progenitor? Perhaps even more interestingly, could one derive new mutant alleles at the appropriate loci, then cross the mutants in such a way as to come up with a new maize? Could one even start with other grasses that have never been domesticated, and reproduce in a few years what took thousands of years in the original successful experiment? The answers are not known, but there is no reason to think that these things cannot be done, and the methods to do them are becoming available. References
1. DOEBLEY J: Molecular evidence and the evolution of maize. Econ Bot 1990, 44(Suppl):6-27. 2. SUNDBERG MD, ORR AR: Inflorescence development in two annual teosintes: Zea mays subsp. mexlcana and Z. mays subsp. parvlglumis. AmerJBot 1990, 77:141-152. 3. LONGLEY AE: Chromosome morphology in maize and its relatives. Bot Rev 1952, 18:399-412
4.
EMERSON RA, BEADLE GW: Studies of Euchlaena and its hybrids
with Zea II. Crossing over between the chromosomes of Euchlaena and those of Zea. Z nduk. Abstamm Verer 1932, 62:305-315. 5. BEADLE GW: The mystery of maize. Field Mus Nat Hist Bull 1972, 43:2-11. 6. DOEBLEY J: Mapping the genes that made maize. Trends Genet 1992, 8:302-307.
7. DOEBLEY J, STEC A: Genetic analysis of the morphological differences between maize and teosinte. Genetics 1991, 129: 8.
285-295. DOEBLEY J, STEC A: Inheritance of the morphological differ-
9.
two F2 populations. Genetics 1993, 134:559-570. DORWEILER J, STEC A, KERMICLE J, DOEBLEY J: Teosinte glume
ences between maize and teosinte: comparison of results for architecture 1: A genetic locus controlling a key step in maize evolution. Science 1993, 262:233-235. 10. BEADLE GW: Teosinte and the origin of maize. J Hered 1939, 30:245-247.
Elliot M. Meyerowitz, Division of Biology 156-29, California Institute of Technology, Pasadena, California 91125, USA.
IN THE APRIL 1994 ISSUE OF CURRENT OPINION IN BIOTECHNOLOGY Nam-hai Chua will edit the following reviews on Plant Biotechnology: Manipulation of carbohydrate partitioning by M. Stitt Novel strategies for engineering virus resistance by D. Baulcombe Gene expression in transgenic monocotyledons by K. Shimamoto Disease resistance results from phytoalexin expression by R. Hain Control of ripening by A. Theologis The role of salicylic acid in systemic acquired resistance by D. Horvath and N-H. Chua Lipid modification by A. Kinney The same issue will also include the following reviews on Biochemical Engineering, which will be edited by Sheldon May and Robert Schwartz: Large-scale mammalian cell culture by M. Reiter Large-scale insect and plant cell culture by M. Schuler Metabolic engineering by G. Stephanopoulos Large-scale processing of macromolecules by S. Fulton Fermentation monitoring and process control by T. Scheper New applications of biocatalysts by S. Neidleman
PLANT EVOLUTION
De-evolution and re-evolution of maize Studies of the genetic differences between maize and its wild predecessor have started to reveal genes responsible for evolutionary innovations. Maize was derived by human selection from a wild progenitor called teosinte, a grass that grows in southern Mexico and Guatemala. Both plants are taxonomically termed Zea mays, though they are given different subspecific designations, with maize being Zea mays subsp. mays, and the strain of teosinte most closely related to maize designated Zea mays subsp. parviglumis [1]. Despite their close relationship, maize and teosinte have major structural, and therefore developmental, differences. Elucidation of the molecular basis of these differences could shed interesting light on the evolutionary origin of novel morphological features in plants. Studies with this aim in mind are beginning to bear fruit. Although maize and teosinte closely resemble each other in their stem, leaf and root structures and in their male inflorescences (tassels), there are several important differences between them (Fig. 1). Maize has a single primary stem with a tassel on top, whereas teosinte is branched, and has a tassel not only on the
top of the main stem, but also at the apex of lateral branches. The female inflorescence of maize is the familiar ear, consisting of a cob covered with kernels (fruits containing a single seed). The simplest maize ears are four-ranked, with two kernels per rank due to the development of paired spikelet primordia. They thus have a minimum of eight rows of kernels around the cob circumference. The female inflorescence of teosinte consists of a slender rachis (an ordinary stem, quite unlike a cob) with seeds projecting from it on only two sides. This inflorescence is two-ranked, with only one kernel per rank due to the abortion of one of the two spikelet primordia [2]. Maize kernels are naked on the cob, with only a small, papery chaff surrounding the base of each kernel. Teosinte kernels are covered on one side by outgrowths of rachis tissue, and on the other by a tough and extremely hard glume. They are consequently inaccessible and inedible except when very young, before the glume hardens, or when somehow extracted from
Fig. 1. Comparison of a modern maize plant, maize ear and a pair of maize kernels (left) with a teosinte plant, teosinte ear and teosinte kernels (right). (Adapted from [5].)
©Current Biology 1994, Vol 4 No 2
127
128
Current Biology 1994, Vol 4 No 2 the glume. In addition, maize seeds stay on the plant when mature (a key characteristic of seed crops), whereas the kernels of teosinte, like those of any wild plant, disperse when they are mature. Maize cannot survive without human intervention because of its inability to disperse its seeds, and because of the vulnerability of the naked seeds to insects and rodents. Maize is thus a genetically engineered plant, which lives only as a human symbiont. Despite the remarkable morphological differences between maize and teosinte, we know that they are extremely close relatives. The archeological evidence indicates that native Americans derived maize from teosinte less than 10000 years ago. Both maize and teosinte have a haploid content of ten chromosomes, which have the same lengths and arm ratios, and heterochromatic knobs in the same relative positions 131. Analyses of isozymes and of chloroplast DNA restriction fragments have shown that maize and teosinte are genetically very similar - far more similar to each other than either is to other related plants [1]. The two plants are completely compatible in crosses,
with normal recombination occurring over most or all chromosome intervals [4]. This leaves us with the profound genetic question of how maize was created from teosinte. What sorts of mutations were obtained, and bred together, to create a new plant architecture, the novel structure of the ear and a naked fruit? That is, what is the nature of the genetic differences between the two close relatives that leads them to have such different patterns of development? This is a restatement of a central question in evolution - what is the molecular and genetic mechanism by which morphological innovation occurs? The genetic basis of the origin of maize was first successfully approached by Beadle, who crossed a primitive race of maize to teosinte and then self fertilized the F1 hybrid progeny. In a large population of the resulting F2 progeny, Beadle found that the parental phenotypes reappeared at a frequency of around 1/500, indicating that the differences between maize and teosinte might be due to as few as five major unlinked Mendelian loci [5]. Earlier (and subsequent) crosses between modern agricultural maize varieties and teosinte gave far more complex results, probably because there are at least as many genetic differences between primitive, small-eared varieties of maize and their enormous modern descendants as there are between teosinte and the primitive maize races [6]. Beadle's experiment could not, however, identify the genetic map locations of the genes that made teosinte into maize, because the genetic marker lines of maize have the background of modern varieties. In addition, several different genetic regions contribute to certain of the single morphological differences between teosinte and maize, making a Mendelian analysis of the differences difficult. The problems of inadequate genetic markers in primitive maize, and of multigenic inheritance, have now been solved and Beadle's analysis repeated. The problem of genetic markers for the mapping of differences in teosinte and maize has been solved by the use of restriction fragment length polymorphism (RFLP) markers. Teosinte and maize (and different maize varieties) show abundant RFLPs, which have been mapped across the entire genome. The problem of analyzing multigenic inheritance has been solved by application of new statistical methods for quantitative trait locus (QTL) mapping. Doebley and his co-workers [7,8] have used RFLPs and QTL mapping to define and genetically map the differences between teosinte and a primitive maize variety, and have found that, consistent with Beadle's result, five major QTLs control most of the morphological differences between teosinte and its descendant.
Fig. 2. Photograph of a teosinte rachis with its two rows of kernels (left) next to the rachis of a teosinte plant into which the maize Tgal allele has been introgressed (right). Note the reduction in the cupule and the outward growth of the glume caused by the maize gene. (Photograph courtesy of John Doebley.)
The difference between the single stem and single tassel of maize, and the branched architecture and multiple tassels of teosinte, is largely controlled by a small region on chromosome arm 1L, the maize copy of which dominantly, or co-dominantly, confers the maize
DISPATCH
branching pattern. There is a known recessive genetic mutation in modern maize that mimics the teosinte condition, teosinte branched (tbl), and this maps to the same chromosome arm as the branching pattern determinant. Most of the difference between the soft glumes of maize and the extremely hard glumes of teosinte correlates with a genomic region near the centromere on the short arm of chromosome 4. Again, the maize genetic region acts dominantly or co-dominantly to the homologous teosinte region. The difference between the two-ranked teosinte female inflorescence and the many-ranked maize ear depends on the paired floral spikelets of maize (teosinte also makes paired spikelet primordia, but one member of each pair aborts), as well as on there being more ranks of spikelet pairs in maize. The paired spikelets map predominantly to locations on chromosomes 1 and 3L, whereas the inflorescence phyllotaxy (rank) depends to a large degree on a portion of chromosome arm 2S, and a region on chromosome 5. The ease with which teosinte kernels fall from the plant depends on a region of chromosome arm 1L, with a contribution from other regions that may vary depending on the race of primitive maize used in the crosses (this character may thus have independently evolved from the teosinte condition in different maize lines). Five genomic regions are therefore responsible for the greatest part of the striking morphological differences between teosinte and maize. Do each of these regions represent single genes? What is the phenotypic effect of each of these regions alone - that is, what does teosinte look like with each of the single maize chromosomal loci, and conversely, what does maize look like with each of the single teosinte loci? The answer to these questions has now been obtained [9] for the chromosome 4 region that is largely responsible for the small and soft glumes of maize. Using RFLPs to speed the process, the teosinte version of this region has been introgressed into a maize genetic background, and vice versa. Analysis of the resulting near-isogenic lines has demonstrated that the chromosome 4S region contains a single gene affecting the glume. This gene has been named teosinte glume architecture 1 (tgal). The maize allele (Tgal) in a teosinte background makes the cupule - the indented part of the rachis that covers one side of the seed shallow, and makes the glume that normally covers the other side of the seed point outward at maturity, rather than curving over the seed (Fig. 2). The net effect is that the seed angles out from the rachis, and is partially exposed. In the converse strain, a de-evolved maize variety with the teosinte (tgal) allele in a maize background, the normally small and soft glume of maize is harder and thicker, and grows in a way that causes it to curve over, and partly cover, the kernel (Fig. 3). This work for the first time reveals the genetic nature and phenotypic effect of a single one of the small number of genes responsible for the evolution of maize from teosinte, and shows how the individual effects of the other
Fig. 3. Comparison of maize ears (without kernels). On the left is an ear homozygous for the maize Tgal allele; the ear on the right is homozygous for the teosinte tga I allele. The outer glumes of the ear on the left are thin and perpendicular to the ear axis, whereas those of the ear on the right are thick and curved upward. (Photograph courtesy of John Doebley.)
genes can also be elucidated. The molecular cloning of Tgal is now possible, and should soon reveal the nature of the gene product and of the mutation that distinguishes the teosinte and maize alleles. The Tgal results also shed light on an ethnobotanical mystery: why would people start to grow and breed something as useless as teosinte seems to be? As Kempton put it in 1937, "No more useless grasses from the standpoint of human consumption could be devised than the American relatives of maize" (quoted in [10]). We can consider two hypotheses. One is that there is a use for teosinte that would give a reason to cultivate the plant. Beadle [10] reported that he could pop the kernels of teosinte to make popcorn. The kernels might thus have been desirable enough to serve as encouragement for propagation, at least if the early Mexicans were, like Beadle, extraordinarily fond of popcorn (N. Horowitz, personal communication). Beadle [5] later reported experiments in which he ground and ate mature teosinte kernels and did not get sick. An alternative hypothesis is that the use of teosinte as food came only after one or more of the mutations that separate maize and teosinte were present in a local teosinte population. Tgal is an excellent candidate for such a gene: this allele alone, in a genetic background that is otherwise that of teosinte, markedly alters the glumes and, by changing the shape of cupules and glumes, exposes the seeds. Tgal mutant teosinte may thus have been the original crop, to which the other mutations were later added. One question raised by the recent genetic studies of teosinte is whether we can soon re-evolve maize. Could one find populations of teosinte that are segregating for
129
130
Current Biology 1994, Vol 4 No 2 the mutant alleles that have been fixed to create maize? Could one use RFLP markers to identify plants carrying the mutant alleles, which could be crossed to reconstitute maize from its progenitor? Perhaps even more interestingly, could one derive new mutant alleles at the appropriate loci, then cross the mutants in such a way as to come up with a new maize? Could one even start with other grasses that have never been domesticated, and reproduce in a few years what took thousands of years in the original successful experiment? The answers are not known, but there is no reason to think that these things cannot be done, and the methods to do them are becoming available. References
1. DOEBLEY J: Molecular evidence and the evolution of maize. Econ Bot 1990, 44(Suppl):6-27. 2. SUNDBERG MD, ORR AR: Inflorescence development in two annual teosintes: Zea mays subsp. mexlcana and Z. mays subsp. parvlglumis. AmerJBot 1990, 77:141-152. 3. LONGLEY AE: Chromosome morphology in maize and its relatives. Bot Rev 1952, 18:399-412
4.
EMERSON RA, BEADLE GW: Studies of Euchlaena and its hybrids
with Zea II. Crossing over between the chromosomes of Euchlaena and those of Zea. Z nduk. Abstamm Verer 1932, 62:305-315. 5. BEADLE GW: The mystery of maize. Field Mus Nat Hist Bull 1972, 43:2-11. 6. DOEBLEY J: Mapping the genes that made maize. Trends Genet 1992, 8:302-307.
7. DOEBLEY J, STEC A: Genetic analysis of the morphological differences between maize and teosinte. Genetics 1991, 129: 8.
285-295. DOEBLEY J, STEC A: Inheritance of the morphological differ-
9.
two F2 populations. Genetics 1993, 134:559-570. DORWEILER J, STEC A, KERMICLE J, DOEBLEY J: Teosinte glume
ences between maize and teosinte: comparison of results for architecture 1: A genetic locus controlling a key step in maize evolution. Science 1993, 262:233-235. 10. BEADLE GW: Teosinte and the origin of maize. J Hered 1939, 30:245-247.
Elliot M. Meyerowitz, Division of Biology 156-29, California Institute of Technology, Pasadena, California 91125, USA.
IN THE APRIL 1994 ISSUE OF CURRENT OPINION IN BIOTECHNOLOGY Nam-hai Chua will edit the following reviews on Plant Biotechnology: Manipulation of carbohydrate partitioning by M. Stitt Novel strategies for engineering virus resistance by D. Baulcombe Gene expression in transgenic monocotyledons by K. Shimamoto Disease resistance results from phytoalexin expression by R. Hain Control of ripening by A. Theologis The role of salicylic acid in systemic acquired resistance by D. Horvath and N-H. Chua Lipid modification by A. Kinney The same issue will also include the following reviews on Biochemical Engineering, which will be edited by Sheldon May and Robert Schwartz: Large-scale mammalian cell culture by M. Reiter Large-scale insect and plant cell culture by M. Schuler Metabolic engineering by G. Stephanopoulos Large-scale processing of macromolecules by S. Fulton Fermentation monitoring and process control by T. Scheper New applications of biocatalysts by S. Neidleman