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What causes change of sex in certain monoecious plants such as Papaya?

What causes change of sex in certain monoecious plants such as Papaya?


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certain male flowering plants can be converted to female flowering and fruiting such as Papaya plants by just insertion of an iron nail in their/it's stem near it's roots? How can iron nail alter it genetically from male to female? Biochemically what causes change in sex of plants from male to female?


Structural variations in papaya genomes

Structural variations (SVs) are a type of mutations that have not been widely detected in plant genomes and studies in animals have shown their role in the process of domestication. An in-depth study of SVs will help us to further understand the impact of SVs on the phenotype and environmental adaptability during papaya domestication and provide genomic resources for the development of molecular markers.

Results

We detected a total of 8083 SVs, including 5260 deletions, 552 tandem duplications and 2271 insertions with deletion being the predominant, indicating the universality of deletion in the evolution of papaya genome. The distribution of these SVs is non-random in each chromosome. A total of 1794 genes overlaps with SV, of which 1350 genes are expressed in at least one tissue. The weighted correlation network analysis (WGCNA) of these expressed genes reveals co-expression relationship between SVs-genes and different tissues, and functional enrichment analysis shows their role in biological growth and environmental responses. We also identified some domesticated SVs genes related to environmental adaptability, sexual reproduction, and important agronomic traits during the domestication of papaya. Analysis of artificially selected copy number variant genes (CNV-genes) also revealed genes associated with plant growth and environmental stress.

Conclusions

SVs played an indispensable role in the process of papaya domestication, especially in the reproduction traits of hermaphrodite plants. The detection of genome-wide SVs and CNV-genes between cultivated gynodioecious populations and wild dioecious populations provides a reference for further understanding of the evolution process from male to hermaphrodite in papaya.


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II. Sex chromosomes and estimation of their ages from sequence divergence

I define plant sex chromosomes as genome regions of these species that carry the ‘SEX’ locus that controls the sexes of individuals, and that do not recombine. Rather than using the term sex chromosomes, I shall often use ‘fully sex-linked regions’ (and full sex linkage), because of the diversity among plants with genetically controlled dioecy – some have extensive non-pairing regions that show heteromorphism between the sexes, like many animal sex chromosomes, but many have no detectable cytological differences (recently reviewed by Renner, 2014 ). Silene latifolia is an example of sex chromosome heteromorphism. It has an XY system, and males are the heterozygous sex, as in mammals (Bellott et al., 2014 Cortez et al., 2014 ). The Y is largely non-recombining, with XY pairing only in a small pseudo-autosomal region (PAR) at one tip (Westergaard, 1958 Filatov et al., 2008 ) mapping of genic markers suggests a single PAR (Bergero et al., 2013 ), although an amplified fragment length polymorphism (AFLP) map suggests two (Scotti & Delph, 2006 ). However, unlike many animal Y chromosomes, the fully sex-linked region still carries hundreds of genes (Bergero & Charlesworth, 2011 Chibalina & Filatov, 2011 Muyle et al., 2012 ). By contrast, the fully Y-linked region in papaya is only c. 10% of chromosome 1 (Liu et al., 2004 Wang et al., 2012 ). Some diploid plants have ZW systems, in which females are ZW heterozygotes and males are ZZ homozygotes (Westergaard, 1958 ), as in birds (Zhou et al., 2014 ) and Lepidoptera (Suetsugu et al., 2013 ) these include Fragaria (strawberry) species (Spigler et al., 2008 Goldberg et al., 2010 ) and Salix (Alstrom-Rapaport et al., 1998 ). Other systems, including those in haploid plants, will be described below.

The time at which recombination stopped can be estimated using DNA sequence divergence between genes present on the Y as well as the X chromosome, together with a ‘molecular clock’ for synonymous or silent site divergence per year. Higher X–Y divergence values correspond to greater times since recombination suppression. In both humans (Lahn & Page, 1999 ) and the plant S. latifolia (Bergero et al., 2007 ), divergence increases with the distance from the PAR (in X chromosome genetic or physical maps as these Y chromosomes are extensively rearranged, distances on the Y chromosome are not informative Skaletsky et al., 2003 Bergero et al., 2008 ). Therefore, suppressed recombination must have spread from an early non-recombining region, the oldest ‘evolutionary stratum’ (Lahn & Page, 1999 ), towards younger ‘strata’ closer to the current PAR. X–Y divergence in the older S. latifolia stratum is similar to that in the youngest of the five strata in humans (Skaletsky et al., 2003 ), and the Silene XY pair probably evolved c. 5–10 million yr ago (MYA Nicolas et al., 2005 ).

A sex chromosome system may be older than its oldest stratum, because recombination suppression in a sex-determining region usually takes time to evolve (Section VIII). However, recombination suppression may pre-date the evolution of separate sexes. In several well-studied plants, results from the combination of genetic and physical mapping reveal large genome regions with infrequent crossing over the surrounding centromeres, with crossovers restricted to the ends of chromosomes, for example, in maize (Rodgers-Melnicka et al., 2015 ). These regions may include substantial proportions of genes in barley, for example, c. 20% of genes are estimated to be located in such regions (Baker et al., 2014 ). If sex-determining loci evolve in such a region, the oldest stratum will be contemporaneous with the sex-determination system (Fig. 1).

In what follows, I stress the importance of estimating the ages of non-recombining regions in order to understand several important aspects of sex chromosome evolution. Young sex chromosome systems are well suited for the study of the early stages of evolution of recombination suppression and the evolution of these characteristics in older animal systems, these processes can only be studied over a coarse timescale that cannot reveal much detail. Young evolutionary strata in plant sex chromosomes are also of interest for the study of the time course of genetic degeneration, including gene losses from Y chromosomes.


Methods

V. monoica BAC Library Screening and DNA Isolation.

The V. monoica BAC library was screened following the protocol of the DIG High Primer DNA Labeling and Detection Starter Kit II (Roche), using probes designed from papaya X BACs located throughout the X-linked region and a probe designed from a papaya autosomal BAC. Twelve positive BACs were confirmed using PCR, 11 corresponding to the X-linked region, and 1 corresponding to the papaya autosomal region (SI Appendix, Fig. S4). A miniprep BAC DNA isolation was performed to check the insert size of each BAC via clamped homogeneous electric field gel electrophoresis. The BAC-carrying cells were grown at 37 °C overnight using glycerol stock from a single colony and isolated using the BACMAX DNA purification kit from Epicenter Biotechnologies (cat# BMAX044).

RNA Isolation.

RNA for RT-PCR (see below) was isolated from V. monoica leaves and flowers using the phenol/chloroform method. After testing the RNA quality using gel electrophoresis, the RNA was treated with DNase, and reverse transcribed into cDNA using ImProm-II Reverse Transcription System from Promega (Cat# A3800).

BAC Sequencing.

Eleven V. monoica BACs (∼1.10 Mb) corresponding to the X-linked region of the papaya X chromosome (∼2.56 Mb) and one V. monoica BAC (∼100 kb) corresponding to a papaya autosomal region (∼72.8 kb) were sequenced, using Sanger and 454 sequencing technology, and assembled, using Roche’s GS assembler, leaving only a few gaps in the sequences. BAC sequences are available through the National Center for Biotechnology Information (NCBI) (SI Appendix, Table S11).

C. papaya and V. monoica Alignments.

The 11 V. monoica BACs, as well as the corresponding 16 BACs and two contigs of the papaya X-linked region, were combined to make V. monoica and papaya pseudomolecules and a synteny analysis and dot-plot comparison were performed using Symap with the default settings (30). Chromosome expansion and collinearity analyses were performed using the genome alignment tool Mauve with the default settings (31). Sequence comparisons between the papaya X-linked pseudomolecule and the corresponding V. monoica pseudomolecule, as well as the papaya and V. monoica autosomal BACs, were carried out using the Artemis Comparison Tool developed by the Sanger Institute with a 500-bp alignment length.

C. papaya and V. monoica Repeat Analysis.

To annotate repetitive sequence, a combination of TEdenovo from the REPET pipeline (32), RepeatScout (33) and Recon1.05 (34) repeat annotation software were used to identify novel V. monoica-specific repetitive elements. Eleven BACs from V. monoica corresponding to the X-linked region in papaya, as well as one autosomal BAC and whole-genome shotgun assemblies of V. monoica genomic DNA, were used to create a custom repeat dataset. Redundancies in the dataset were eliminated using CD-HIT software (35). V. monoica specific repeats were combined with Repbase (36), TIGR plant repeats (http://plantrepeats.plantbiology.msu.edu/index.html), and papaya-specific repeats (37) to generate a custom library. This nonredundant library was used with RepeatMasker to mask repeats in the 12 V. monoica BACS. A strict cut-off value of 350 was used to ensure that only true repetitive elements were masked. Repetitive elements for the papaya whole genome and X region were taken from Ming et al. and Wang et al., respectively (16, 17). Given the small sample size of V. monoica BACs and low copy number of some repeats, the reported repeat percentages are likely to underestimate the true values.

V. monoica Gene Prediction.

Genes were predicted in the V. monoica BACs using Genscan (http://genes.mit.edu/GENSCAN.html) and FGENESH (http://linux1.softberry.com/berry.phtml), as well as homology to papaya-expressed sequence tags (ESTs) and gene models. The papaya autosomal and X BACs were previously annotated by refs. 17 and 38. V. monoica genes were confirmed through RT-PCR, with primers designed using primer3 (http://frodo.wi.mit.edu/primer3/) to span at least one intron, if possible. V. monoica leaf and flower cDNA were synthesized using Promega ImProm-II Reverse Transcription System (Cat.# A3800). The PCR products were sequenced using Sanger sequencing and manually edited in Sequencher 4.1.10 (Gene Codes Corporation, 2011). The confirmed genes were blasted to the NCBI nonredundant protein database to predict the gene structures and functions through homology. Each individual transcript was translated, and those with premature stop codons were classified as pseudogenes.

Ka/Ks and Divergence Time Analysis.

V. monoica and papaya gene pairs were manually annotated for exon and intron regions using EST sequences, RT-PCR, and homology with genes in the NCBI database. The sequences were aligned using SeaView v4 (39) and exported into DnaSP v5 (40) to estimate synonymous substitutions per synonymous site (Ks), nonsynonymous substitutions per nonsynonymous site (Ka), and synonymous and noncoding plus synonymous substitutions were used to estimate substitutions per silent site (Ksil) using Nei and Gojobori’s method (41). Divergence times were calculated using the Ksil values, calibrated with the synonymous substitution rate of 4 × 10 −9 substitutions per synonymous site per year determined for Arabidopsis, a member of Brassicacea, the closest family to Caricaceae (42). CpXY h 20, CpXY h 29, and CpXY h 37 were removed from the divergence time analysis because of missing sequence data (incomplete BAC sequences).

V. monoica Genome Survey Sequencing.

The V. monoica whole genome was survey-sequenced using one lane of Illumina sequencing and the sequences are available at (http://www.life.illinois.edu/ming/LabWebPage/Downloads.html).


↵ 1 J.W., J.-K.N., Q.Y., and A.R.G. contributed equally to this work.

↵ 2 Present address: Department of Agronomy, University of Florida, Gainesville, FL 32610.

↵ 3 Present address: Department of Molecular Breeding, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-701, Republic of Korea.

↵ 4 Present address: Departamento de Genética, Facultad de Ciencias, Universidad de Granada Campus de Fuentenueva Sin Numero, 18071 Granada, Spain.

↵ 5 Present address: Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634.

↵ 6 Present address: Department of Agronomy, Shandong Agricultural University, Tai’an, Shandong 271018, China.

Author contributions: Q.Y., M.A., P.H.M., J.J., A.H.P., and R.M. designed research J.W., J.-K.N., Q.Y., A.R.G., J.H., F.Z., R.A., R.V., J.E.M., W.Z., R.N.-P., F.A.F., C.L., E.J.T., C.C., C.M.W., R.S., M.-L.W., X.J.M., J.J., and R.M. performed research J.W., J.-K.N., Q.Y., A.R.G., J.H., F.Z., R.A., R.V., J.E.M., W.Z., R.N.-P., F.A.F., C.L., E.J.T., C.C., C.M.W., R.S., M.-L.W., X.J.M., M.A., D.C., P.H.M., J.J., A.H.P., and R.M. analyzed data and J.W., A.R.G., D.C., and R.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. R.G.K. is a guest editor invited by the Editorial Board.

Database deposition: The sequences reported in this paper have been deposited in the GenBank database. See SI Appendix, Table S14 for accession nos.


THE HOMOSPOROUS FERNS

Recent phylogenetic analyses of vascular seed–free plants group the leptosporangiate and eusporangiate ferns and members of Equisetum and Psilotum into a monophyletic clade that is sister to the seed plants ( Pryer et al., 2001). With few exceptions, they are homosporous plants. The one plant for which a sex-determining pathway has been genetically well defined is the leptosporangiate fern Ceratopteris richardii. Like Marchantia, Ceratopteris is homosporous and produces only one type of haploid spore. Although the sex of the Marchantia gametophyte is determined genetically by sex chromosomes, the sex of the Ceratopteris gametophyte (male or hermaphroditic) is determined epigenetically by the pheromone antheridiogen. Since their discovery by Dopp (1950)in the fern Pteridium aquilinum, antheridiogens have been identified and characterized from many species of leptosporangiate ferns (reviewed by Naf, 1979 Yamane, 1998), suggesting that it is a common mode of regulating sexual phenotypes in this group of plants. Although the structure of the Ceratopteris antheridiogen is unknown, all other fern antheridiogens characterized to date are mostly novel gibberellins ( Yamane, 1998).

The Sex-Determining Mutants of Ceratopteris richardii.

The her1 (hermaphroditic) mutant and the wild-type hermaphrodite are indistinguishable, as are the tra1 (transformer) mutant and the wild-type males, except that the her1 and tra1 mutants are insensitive to the absence or presence of ACE. The ACE-insensitive fem1 (feminization) gametophyte produces no antheridia. The man1 (many antheridia) mutant produces ∼10 times more antheridia than hermaphrodites, whereas the not1 (notchless) mutant rarely produces antheridia. The meristem notch normally present on the hermaphrodite often is missing in the not1 mutant, giving it a cup-shaped appearance. The novel phenotypes of the fem1 tra1 and fem1 not1 tra1 mutants are shown. an, antheridia ar, archegonia mn, meristem notch.

The Sex-Determining Mutants of Ceratopteris richardii.

The her1 (hermaphroditic) mutant and the wild-type hermaphrodite are indistinguishable, as are the tra1 (transformer) mutant and the wild-type males, except that the her1 and tra1 mutants are insensitive to the absence or presence of ACE. The ACE-insensitive fem1 (feminization) gametophyte produces no antheridia. The man1 (many antheridia) mutant produces ∼10 times more antheridia than hermaphrodites, whereas the not1 (notchless) mutant rarely produces antheridia. The meristem notch normally present on the hermaphrodite often is missing in the not1 mutant, giving it a cup-shaped appearance. The novel phenotypes of the fem1 tra1 and fem1 not1 tra1 mutants are shown. an, antheridia ar, archegonia mn, meristem notch.

The Genetic Sex-Determining Pathways in the Fern Ceratopteris, the Fly Drosophila melanogaster, and the Nematode Caenorhabditis elegans.

The genetic model of sex determination in Ceratopteris ( Strain et al., 2001) is dependent on two genes, FEM1 and TRA (there are at least two TRA genes), which promote the differentiation of male (antheridia) and female (meristem and archegonia) traits, respectively. FEM1 and TRA also antagonize each other such that if FEM1 is active, TRA is not, and vice versa. What determines which of these two genes prevails in the gametophyte and thus its sex is the pheromone ACE, which activates the HER genes, of which there are at least five, and sets into motion a series of switches that ultimately result in male development (i.e., FEM1 on and TRA off). These switches are thrown in the opposite direction when spores germinate in the absence of ACE. Although FEM1 represses TRA and TRA represses FEM1, they do not do so directly. TRA activates MAN1, which represses FEM1, and FEM1 activates NOT1, which represses TRA. Because TRA and FEM1 are the primary regulators of sex, NOT1 and MAN1 are considered regulators of the regulators. The sex determination pathway in C. elegans ( Hodgkin, 1987 Villeneuve and Meyer, 1990) is linear and consists of a series of negative control switches. The state of the initial switch gene (xol-1) is dependent on the ratio of X to autosomal (A) chromosomes. If the ultimate downstream gene in this pathway (TRA-1) is high, the nematode develops as a hermaphrodite. If TRA-1 is low, it develops as a male. The linear sex determination pathway shown for Drosophila is from the mid-1980s ( Baker and Ridge, 1980 Cline, 1983). There are actually other sex-determining pathways that account for most aspects of sexual phenotype (reviewed by Oliver, 2002) the pathway shown is the somatic pathway. In the soma, Sxl is the key regulator of sex, and its state of activity is determined by the X:A ratio. The dsx gene is the downstream regulatory gene that ultimately determines whether male or female genes are expressed in the soma.

The Genetic Sex-Determining Pathways in the Fern Ceratopteris, the Fly Drosophila melanogaster, and the Nematode Caenorhabditis elegans.

The genetic model of sex determination in Ceratopteris ( Strain et al., 2001) is dependent on two genes, FEM1 and TRA (there are at least two TRA genes), which promote the differentiation of male (antheridia) and female (meristem and archegonia) traits, respectively. FEM1 and TRA also antagonize each other such that if FEM1 is active, TRA is not, and vice versa. What determines which of these two genes prevails in the gametophyte and thus its sex is the pheromone ACE, which activates the HER genes, of which there are at least five, and sets into motion a series of switches that ultimately result in male development (i.e., FEM1 on and TRA off). These switches are thrown in the opposite direction when spores germinate in the absence of ACE. Although FEM1 represses TRA and TRA represses FEM1, they do not do so directly. TRA activates MAN1, which represses FEM1, and FEM1 activates NOT1, which represses TRA. Because TRA and FEM1 are the primary regulators of sex, NOT1 and MAN1 are considered regulators of the regulators. The sex determination pathway in C. elegans ( Hodgkin, 1987 Villeneuve and Meyer, 1990) is linear and consists of a series of negative control switches. The state of the initial switch gene (xol-1) is dependent on the ratio of X to autosomal (A) chromosomes. If the ultimate downstream gene in this pathway (TRA-1) is high, the nematode develops as a hermaphrodite. If TRA-1 is low, it develops as a male. The linear sex determination pathway shown for Drosophila is from the mid-1980s ( Baker and Ridge, 1980 Cline, 1983). There are actually other sex-determining pathways that account for most aspects of sexual phenotype (reviewed by Oliver, 2002) the pathway shown is the somatic pathway. In the soma, Sxl is the key regulator of sex, and its state of activity is determined by the X:A ratio. The dsx gene is the downstream regulatory gene that ultimately determines whether male or female genes are expressed in the soma.

In comparing mechanisms of gametophytic sex determination in homosporous bryophytes and ferns, one obvious question that arises is what drove Marchantia to an X-Y chromosomal mechanism of sex determination and Ceratopteris to an epigenetically regulated mechanism dependent on pheromonal cross-talk between individuals? The answer to this question probably lies in the different ratios of males and females or hermaphrodites that occur in the populations of each species. In Marchantia, the segregation of X and Y sex chromosomes during meiosis in the sporophyte ensures that each gametophyte progeny has an equal probability of being either male or female, barring selection. In Ceratopteris, the ACE response allows the ratio of males to hermaphrodites to vary depending on the density of the population, such that as the population density increases, the proportion of males also increases. Although the underlying sex-determining mechanism is inflexible in Marchantia, it is flexible enough in Ceratopteris to allow each individual to determine its sex according to the size of the population in which it resides and the speed at which it germinates relative to its neighbors. The flexibility of the Ceratopteris sex-determining mechanism is reflected in its sex-determining pathway, and this becomes especially apparent compared with the sex-determining pathways known from other organisms, including Drosophila melanogaster and Caenorhabditis elegans, which are illustrated in Figure 3. In both of these animals, an individual's sex (male or female in D. melanogaster and male or hermaphrodite in C. elegans) is determined genetically by the ratio of X to autosomal chromosomes. This ratio is read and either activates or represses the activities of downstream genes in each pathway. In both animals, the sex ultimately depends on the state of the terminal gene in each linear pathway, TRA1 in the case of C. elegans. The Ceratopteris sex-determining pathway is distinctly different from those of D. melanogaster and C. elegans in that it is not linear and there are two sex-determining genes, one for male and one for female development. Their ability to repress each other endows each Ceratopteris spore with the flexibility to determine its sex upon germination based on environmental cues.

So why would a flexible mechanism of sex determination that allows sex ratios to vary be adaptive in ferns but not in bryophytes? The answer to this question may lie in the ephemeral nature of the fern gametophyte. Although the gametophytes of bryophytes are persistent, the gametophytes of ferns are not. In Ceratopteris, for example, gametophytes reach sexual maturity only 14 days after spore inoculation and die once they are fertilized. The limited time that a fern gametophyte is able to be crossed by another might favor a sex-determining system that would promote outcrossing by increasing the proportion of males when population densities are high and ensuring a high proportion of hermaphrodites capable of self-fertilization when population densities are low. Because there are a variety of sex-determining mutants available in Ceratopteris, the hypothesis related to the consequences of variable versus fixed sex ratios can be tested easily, at least under defined laboratory conditions.

Future studies to clone the sex-determining genes in Ceratopteris will be necessary to understand their biochemical functions and to test their interactions predicted by the genetic model. Although the size of the Ceratopteris genome is probably very large (n = 37), the ability to inactivate genes in the Ceratopteris gametophyte by RNA interference ( Stout et al., 2003 G. Rutherford, M. Tanurdzic, and J.A. Banks, unpublished observations) provides an important means to study the effects of inactivating potential sex-determining genes in the Ceratopteris gametophyte.


RESULTS

Macroscopic and Microscopic Analyses of the Cucumber Flower

Male cucumber flowers are composed of four whorls of organs (from the outer to the inner whorl): five sepals, five yellow petals, five stamens, and three arrested carpel primordia in the fourth whorl ( Figures 1A to 1C). In contrast, stamens are arrested in their development in the female flower, and the three carpel primordia develop further to an inferior ovary, a short style, and three separated stigmas ( Figures 1D to 1F). To identify stamen primordia in female flowers, we made histological sections of young flower buds. These sections show arrested stamens in the third floral whorl ( Figures 1D and 1E).

Isolation and Expression Analysis of Cucumber Class B and C MADS Box Genes

Due to the high level of sequence conservation within the MADS box domain, MADS box genes have been isolated from various species by using heterologous probes. In this study, class B and C MADS box genes were isolated from cucumber by screening a cucumber female flower–specific cDNA library by using petunia MADS box genes as a probe. One of them, designated CUCUMBER MADS1 (CUM1 Kater et al., 1998), is very similar in its deduced amino acid sequence and expression pattern to the class C genes AGAMOUS (AG) from Arabidopsis ( Yanofsky et al., 1990) and PLENA (PLE) from snapdragon ( Bradley et al., 1993).

Another MADS box gene that was isolated, designated CUM26 (GenBank accession number AF043255), is most likely the ortholog of the previously characterized class B genes PIS-TILLATA (PI) from Arabidopsis ( Goto and Meyerowitz, 1994), GLOBOSA (GLO) from snapdragon ( Schwarz-Sommer et al., 1992), and FLORAL BINDING PROTEIN 1 (FBP1) from petunia ( Angenent et al., 1992). The deduced protein sequence of CUM26 ( Figure 2A) shows that it has 69, 70, and 71% of its amino acid residues in common with PI, GLO, and FBP1, respectively.

The expression patterns of CUM1 and CUM26 were initially studied by RNA gel blot analysis using RNA extracted from leaves and the various floral organs ( Figure 2B). To avoid cross-hybridization due to sequence homology between MADS box genes, we hybridized the RNA gel blot with probes derived from the divergent 3′ ends of the cDNAs. This experiment showed that CUM1 was expressed in stamens and throughout the pistil in the style, stigma, nectary, and ovary. CUM26 expression was similar to that of other class B genes, restricted to the second and third floral whorls.

The expression profiles of these two MADS box genes were examined in more detail by in situ hybridizations on longitudinal sections of young male and female cucumber flower buds. Figure 3Ashows hybridizing signals in stamens and arrested pistil primordia from young male flowers by using antisense RNA derived from the class C gene CUM1 as a probe. In female flowers, CUM1 mRNA accumulated at a low level in stamen primordia that were arrested in development. In pistils, CUM1 was expressed in the stigma, the ovules, and the placenta, whereas no expression was observed in other parts of the ovary ( Figure 3B). CUM26 was expressed in male flowers in petals and young stamens, which is to be expected from a class B gene ( Figure 3C). Similarly, in a female flower, CUM26 transcripts were detectable in petals and arrested stamen primordia ( Figure 3D). These data demonstrate that the presumed class B and C genes are still expressed in primordia that are arrested in development.

Flower Morphology of Wild-Type Cucumber Plants.

(A) Longitudinal section through male flower buds at two developmental stages. The carpel primordia are arrested in whorl 4. Stamen primordia arise from the flanks of the petals and produce sporogenous tissue.

(B) Longitudinal section through a young wild-type male cucumber flower at a later developmental stage just before opening of the flower. The anthers start to produce pollen.

(C) Macroscopic view of a male flower. A pair of sepals and petals were removed to allow a view inside. The arrested carpel primordia are visible at the bottom of the flower.

(D) Longitudinal section through a female flower bud. Stamen and carpel primordia develop in whorls 3 and 4, respectively. The sepals cover the flower completely.

(E) Longitudinal section through a female flower bud at a later developmental stage. The stamen primordia are arrested, and in the fourth whorl an inferior ovary and superior stigmas develop.

(F) Macroscopic view of a female flower at a stage just before opening of the flower. As in (C), a pair of sepals and petals have been removed to allow a view inside the flower.

The whorl numbers indicate the positions of the floral organs within the flower. O, ovary. Bars in (A), (B), (D), and (E) = 1 mm.

Flower Morphology of Wild-Type Cucumber Plants.

(A) Longitudinal section through male flower buds at two developmental stages. The carpel primordia are arrested in whorl 4. Stamen primordia arise from the flanks of the petals and produce sporogenous tissue.

(B) Longitudinal section through a young wild-type male cucumber flower at a later developmental stage just before opening of the flower. The anthers start to produce pollen.

(C) Macroscopic view of a male flower. A pair of sepals and petals were removed to allow a view inside. The arrested carpel primordia are visible at the bottom of the flower.

(D) Longitudinal section through a female flower bud. Stamen and carpel primordia develop in whorls 3 and 4, respectively. The sepals cover the flower completely.

(E) Longitudinal section through a female flower bud at a later developmental stage. The stamen primordia are arrested, and in the fourth whorl an inferior ovary and superior stigmas develop.

(F) Macroscopic view of a female flower at a stage just before opening of the flower. As in (C), a pair of sepals and petals have been removed to allow a view inside the flower.

The whorl numbers indicate the positions of the floral organs within the flower. O, ovary. Bars in (A), (B), (D), and (E) = 1 mm.

Phenotype and Molecular Analyses of a Class B Homeotic Mutant

We studied the homeotic transformations in a spontaneous recessive cucumber mutant designated green petals (gp). Young male flowers of this gp mutant consisted of two perianth whorls of sepals, and outgrowth of the reproductive organs was arrested ( Figure 4A). The male flower became indeterminate when the flower subsequently aged, resulting in a repetition of sepal whorls and a bushy appearance ( Figure 4B). Histological analysis of these male gp flowers revealed that the indeterminate flower buds developed from the third whorl, whereas the fourth inner whorl was arrested in development ( Figures 4C and 4D). Interestingly, when the mutant was grown at high temperature (>30°C), the morphology of the male flower changed dramatically ( Figures 4E and 4F): sepals were still formed in the first two outer whorls, but its indeterminate character was lost, and carpels instead of stamens were produced in the third whorl. In an older male flower ( Figure 4F), the whorl 3 carpels developed into complete parthenocarpic fruit that were positioned superior to the receptacle. This mutant phenotype shows clearly that female organs can develop in male flowers.

Amino Acid Sequence of CUM26 in the Wild Type and the gp Mutant and Expression Patterns of CUM1 and CUM26.

(A) The CUM26 protein sequence deduced from the longest reading frame of CUM26 cDNA. The conserved MADS box is underlined with a thick line, and the K box region is underlined with a thin line. The 15 amino acid residues that are deleted in the CUM26 protein of the gp mutant are boxed.

(B) CUM1 and CUM26 expression in cucumber leaves and floral organs. Total RNA was isolated from mature leaves (L), sepals (S), petals (P), stamens (St), styles (Sl), stigmas (Sg), nectaries (N), and inferior ovaries (O).

Amino Acid Sequence of CUM26 in the Wild Type and the gp Mutant and Expression Patterns of CUM1 and CUM26.

(A) The CUM26 protein sequence deduced from the longest reading frame of CUM26 cDNA. The conserved MADS box is underlined with a thick line, and the K box region is underlined with a thin line. The 15 amino acid residues that are deleted in the CUM26 protein of the gp mutant are boxed.

(B) CUM1 and CUM26 expression in cucumber leaves and floral organs. Total RNA was isolated from mature leaves (L), sepals (S), petals (P), stamens (St), styles (Sl), stigmas (Sg), nectaries (N), and inferior ovaries (O).

Analysis of longitudinal sections of these male flowers at different stages of development indicated that the whorl 4 primordia were also arrested in growth at high temperature. Furthermore, these microscopic analyses confirmed that the superior carpels originated from the third whorl, whereas whorl 4 primordia were still arrested in development ( Figures 4H and 4I). The homeotic conversions observed at high temperature resembled exactly those of class B mutants in species with bisexual flowers, such as Arabidopsis, snapdragon, and petunia ( Coen and Meyerowitz, 1991 Angenent et al., 1993). The female flowers, which can be easily recognized by their inferior ovary, produced sepals in both outer whorls, and no obvious differences in whorl 3 and 4 organs were observed between flowers from the gp mutant and wild-type plants ( Figures 4G and 4J). In contrast with the male flowers, the female flowers were not sensitive to different temperature conditions.

To investigate the molecular nature of this gp mutant, we cloned the CUM26 coding sequence by using reverse transcription–polymerase chain reaction (PCR) on RNA from very young gp flower buds. Sequence analysis demonstrated that this gene contains an in-frame deletion of 15 amino acids just downstream from the region encoding the K domain ( Figure 2A), a motif shown to be involved in protein–protein interactions between MADS box proteins ( Davies and Schwarz-Sommer, 1994 Davies et al., 1996 Fan et al., 1997). For DEFICIENS (DEF), a class B gene of snapdragon, and AG, the class C gene of Arabidopsis, it has been shown that mutations in the K domain lead to temperature-sensitive phenotypes ( Sieburth et al., 1995 Zachgo et al., 1995). Apparently, the deletion in CUM26 affects the function of the protein at high temperatures, resulting in the gp mutant phenotype.

The gp mutant phenotype is linked to this deletion in the CUM26 protein, as demonstrated in a population segregating for the mutant and wild-type phenotypes. In all mutants analyzed (eight plants), the deletion was confirmed by PCR analysis, whereas in the plants with the wild-type phenotype (18 plants), either only the wild-type or both the wild-type and mutant alleles were present (data not shown).

Expression Analysis of CUM1 and CUM26 in gp Mutant Flowers

To analyze the expression of class B and C MADS box genes in male gp flowers in detail, we performed in situ hybridization analysis. Figures 3E and 3Fshow that CUM26 transcripts were not detectable under normal (22°C) and high-temperature (35°C) conditions in male and female (not shown) gp flowers, indicating that the class B function necessary for normal petal and stamen development was not present in this mutant. Surprisingly, hybridization with a CUM1 antisense probe did not reveal any signal in a gp male flower grown at 22°C when no reproductive organs developed ( Figure 3G). In contrast, the same probe detected CUM1 transcripts in the third and fourth floral whorl primordia of plants grown at high temperature ( Figure 3H). Due to the absence of class B gene expression (CUM26) and the expression of CUM1, whorl 3 primordia have a carpel identity according to the ABC model. This was confirmed at later stages of development by the visible outgrowth of superior carpels in the third whorls of these flowers ( Figures 4E and 4F).

Expression Patterns of CUM1 and CUM26 in Wild-Type and gp Mutant Flowers.

Longitudinal sections were hybridized with digoxigenin-labeled antisense CUM1 ([A], [B], [G], and [H]) or CUM26 ([C], [D], [E], and [F]) RNA. All sections were viewed using dark-field microscopy. The whorl numbers indicate the positions of the floral organs within the flower.

(A) and (C) Young male flower buds from a wild-type plant. The carpel primordia are arrested and the anthers start to produce sporogenous tissue. The bud is completely covered by sepals.

(B) and (D) Young female flower buds from a wild-type plant. The stamen primordia are arrested and in the fourth whorl an inferior ovary and superior stigmas develop.

(E) and (G) Male flowers from a gp plant grown under normal temperature conditions (22°C). The red arrowheads indicate new buds as they appear in these bushy indeterminate flowers (cf. Figure 4B).

(F) and (H) Male flowers from a gp plant grown under high-temperature conditions (35°C). Carpels are formed in whorl 3, and the carpel primordia in the fourth whorl are arrested. The outer two whorls are sepals.

O, ovary. Bar in (A) = 1 mm for (A) through (H).

Expression Patterns of CUM1 and CUM26 in Wild-Type and gp Mutant Flowers.

Longitudinal sections were hybridized with digoxigenin-labeled antisense CUM1 ([A], [B], [G], and [H]) or CUM26 ([C], [D], [E], and [F]) RNA. All sections were viewed using dark-field microscopy. The whorl numbers indicate the positions of the floral organs within the flower.

(A) and (C) Young male flower buds from a wild-type plant. The carpel primordia are arrested and the anthers start to produce sporogenous tissue. The bud is completely covered by sepals.

(B) and (D) Young female flower buds from a wild-type plant. The stamen primordia are arrested and in the fourth whorl an inferior ovary and superior stigmas develop.

(E) and (G) Male flowers from a gp plant grown under normal temperature conditions (22°C). The red arrowheads indicate new buds as they appear in these bushy indeterminate flowers (cf. Figure 4B).

(F) and (H) Male flowers from a gp plant grown under high-temperature conditions (35°C). Carpels are formed in whorl 3, and the carpel primordia in the fourth whorl are arrested. The outer two whorls are sepals.

O, ovary. Bar in (A) = 1 mm for (A) through (H).

Ectopic Expression of CUM1 in Cucumber Induces Reproductive Organ Formation in the First and Second Floral Whorls

It has been demonstrated that Arabidopsis plants overexpressing AG under the control of the cauliflower mosaic virus (CaMV) 35S promoter phenocopy apetala2 mutant plants, confirming the model’s prediction that class A suppresses the class C function in the two outer whorls ( Mizukami and Ma, 1992). Overexpression of CUM1 driven by the CaMV 35S promoter in the hermaphrodite species petunia resulted in similarly severe homeotic transformations of sepals into carpelloid structures and petals into stamens ( Kater et al., 1998). However, in the monoecious species cucumber, reproductive organs were arrested in the two types of flowers, raising the question of whether this phenomenon also occurs when the reproductive organs develop at other positions in the flower. Therefore, the CUM1 overexpression construct initially used for the petunia transformations was introduced in cucumber. Two independent transformants, T340-1 and T340-5, showed the most severe and identical homeotic transformations, and in both plants, the CUM1 gene was expressed ectopically, as confirmed by RNA gel blot analysis (data not shown). Although the severe homeotic transformations affect the floral structure significantly, male and female flowers were easily distinguished, because the female flowers still developed an inferior ovary that is not present in male flowers. In whorl 1, the sepals of both male and female flowers of these lines were transformed into carpelloid structures with stigmatic tissue on top ( Figures 5A to 5E). Petals were reduced in size significantly or completely absent in both male and female flowers. Histological analysis was performed to determine the identities of the chimeric organs in whorl 2. This analysis revealed that antheroid tissues, including pollen grains, develop on top of the whorl 2 organs in both male and female flowers ( Figures 5B, 5C, 5E, and 5F). As shown in Figure 5D, the ovaries of these female flowers were malformed, which might be a secondary effect of the aberrations in the other floral organs. The floral phenotypes of these transgenic plants demonstrate that in the second whorl of female flowers male tissue is allowed to develop, whereas in wild-type flowers male organ formation never occurs. Furthermore, female organs develop in the first whorl of male mutant flowers, converting it from a unisexual to a bisexual cucumber flower.

Flower Morphology of the gp Mutant.

(A) Young male flower of the gp mutant grown at 22°C. The flower is composed of two whorls of sepals only.

(B) Older indeterminate male flower grown at 22°C. New buds develop inside the primary flower, which results in a bushy appearance.

(C) Longitudinal section through young male flower buds grown at 22°C. Initially, the whorl 3 and 4 primordia develop as in the wild type (left bud). At a later developmental stage (right bud), new meristems develop in the third whorl, as indicated by the red arrowhead. The carpel primordia in whorl 4 remain arrested.

(D) Longitudinal section through an older indeterminate male flower grown at 22°C. A new flower bud originating from the third whorl in this bushy flower is indicated by the red arrowhead.

(E) Male flower of the gp mutant grown under high-temperature conditions (35°C) with stamens homeotically transformed into carpels.

(F) Older male flower of the gp mutant grown under high-temperature conditions (35°C) with fruit developing in whorl 3. The whorl 1 and 2 organs are senesced.

(G) Female flower of the gp mutant grown at 22°C. The inferior ovary is not affected, and the outer two whorl organs are sepals. No changes in flower phenotype were observed when the plants were grown under high-temperature conditions (35°C).

(H) Longitudinal section through a young male flower of the gp mutant grown under high-temperature conditions (35°C). Carpelloid structures develop in whorl 3, and carpel primordia are arrested in whorl 4.

(I) Older bud as in (H). Fruit-like bodies develop in the third whorl on positions normally occupied by stamens (cf. Figures 4E and 4F).

(J) Longitudinal section through a female flower bud of the gp mutant. The two inner whorls are like those in wild-type flowers (cf. Figure 1E).

The whorl numbers indicate the positions of the floral organs within the flower. O, ovary. Bars in (C), (D), (H), (I), and (J) = 1 mm.

Flower Morphology of the gp Mutant.

(A) Young male flower of the gp mutant grown at 22°C. The flower is composed of two whorls of sepals only.

(B) Older indeterminate male flower grown at 22°C. New buds develop inside the primary flower, which results in a bushy appearance.

(C) Longitudinal section through young male flower buds grown at 22°C. Initially, the whorl 3 and 4 primordia develop as in the wild type (left bud). At a later developmental stage (right bud), new meristems develop in the third whorl, as indicated by the red arrowhead. The carpel primordia in whorl 4 remain arrested.

(D) Longitudinal section through an older indeterminate male flower grown at 22°C. A new flower bud originating from the third whorl in this bushy flower is indicated by the red arrowhead.

(E) Male flower of the gp mutant grown under high-temperature conditions (35°C) with stamens homeotically transformed into carpels.

(F) Older male flower of the gp mutant grown under high-temperature conditions (35°C) with fruit developing in whorl 3. The whorl 1 and 2 organs are senesced.

(G) Female flower of the gp mutant grown at 22°C. The inferior ovary is not affected, and the outer two whorl organs are sepals. No changes in flower phenotype were observed when the plants were grown under high-temperature conditions (35°C).

(H) Longitudinal section through a young male flower of the gp mutant grown under high-temperature conditions (35°C). Carpelloid structures develop in whorl 3, and carpel primordia are arrested in whorl 4.

(I) Older bud as in (H). Fruit-like bodies develop in the third whorl on positions normally occupied by stamens (cf. Figures 4E and 4F).

(J) Longitudinal section through a female flower bud of the gp mutant. The two inner whorls are like those in wild-type flowers (cf. Figure 1E).

The whorl numbers indicate the positions of the floral organs within the flower. O, ovary. Bars in (C), (D), (H), (I), and (J) = 1 mm.

Downregulation of CUM1 Results in a Class C Mutant Phenotype

In addition to the plants ectopically expressing CUM1, the same transformation experiment revealed three cosuppression plants in which endogenous CUM1 expression was abolished completely, as determined by RNA gel blot hybridization (data not shown). Figures 5G to 5Lshow that the cosuppression phenotypes are similar but distinct from that of the Arabidopsis ag mutant. The loss of AG function in Arabidopsis resulted in homeotic transformations of stamens into petals, and a reiteration of the floral program in the center of these flowers replaced the pistil ( Figure 6 Pruitt et al., 1987). Surprisingly, in the male cucumber flowers of the cosuppression plants, five new floral buds appeared in the third whorl ( Figures 5G and 5H). These new flower buds were arranged as two couples and a separate bud occupying the same positions as stamens in wild-type male flowers. In the center of the mutant flower, the rudimentary carpel primordia were replaced by a new indeterminate flower similar to that seen in the Arabidopsis ag mutant flower.

In female flowers in which CUM1 was cosuppressed, petals developed in the third whorl and the floral program reiterated in the center of the flower, with a small fruit growing inside the primary ovary ( Figure 5K, arrow). A cross-view through a female floral bud demonstrated that the whorl 3 primordia were not arrested and developed into petalloid organs ( Figure 5L). The observation that ovaries still develop in these female flowers suggests that CUM1 expression is not required or redundant for the development of the fruit, which is in agreement with the absence of CUM1 expression in the fruit of wild-type flowers ( Figure 3B).

Flower Morphology of Transgenic Cucumber Plants in Which CUM1 Was Ectopically Expressed or Cosuppressed.

(A) Male flower of a CUM1 ectopically expressing plant (T340-1). Five complete superior carpels develop in whorl 1, which are partly fused at the basis forming an ovary-like structure.

(B) Longitudinal section through a male flower of T340-1. The whorl 1 organs are carpelloid, and the organs in the second whorl are chimeric with petaloid and antheroid tissue. The carpel primordia in whorl 4 are arrested.

(C) Detail of (B) as indicated by the box. Antheroid tissue developing on top of the second whorl organs. Sporogenous tissue with developing pollen is indicated by the arrow.

(D) Female flower of T340-1 with superior carpels in whorl 1 and a malformed ovary in the fourth whorl. In the second whorl, remnants of petal tissue is indicated by an arrow.

(E) Longitudinal section through a female flower of T340-1. Antheroid tissue developing on top of the second whorl organs is indicated in the box. The inner part of the flower is highly malformed and organs are not recognizable.

(F) Detail of (E) as indicated by the box. An arrow shows the developing pollen in the antheroid tissue.

(G) Longitudinal view of a male flower bud of transformant T340-3 in which CUM1 was cosuppressed. Indeterminate floral buds are visible in whorls 3 and 4.

(H) Male flower of transformant T340-3 showing indeterminate floral bud formation in positions normally occupied by stamens. The flower is indeterminate in the center.

(I) Female flower of T340-3, with petal formation in the third whorl. The inferior ovary is not affected in this homeotic mutant.

(J) Detail of (I) showing the petals in whorl 3 and the indeterminacy in the fourth whorl.

(K) Longitudinal view of a female flower bud of T340-3. The small fruit growing inside the primary ovary is indicated by an arrow. The line indicates the plane of the cross-section shown in (L).

(L) Cross-section through a female flower bud of T340-3. The position of the section is indicated in (H). Petals develop in whorl 3 on positions normally occupied by stamens. The whorl 4 structure is the upper part of the small fruit that is growing inside the primary ovary.

The whorl numbers indicate the positions of the floral organs within the flower. S, stigmatic tissue O, ovary. Bars in (B), (E), and (L) = 1 mm.

Flower Morphology of Transgenic Cucumber Plants in Which CUM1 Was Ectopically Expressed or Cosuppressed.

(A) Male flower of a CUM1 ectopically expressing plant (T340-1). Five complete superior carpels develop in whorl 1, which are partly fused at the basis forming an ovary-like structure.

(B) Longitudinal section through a male flower of T340-1. The whorl 1 organs are carpelloid, and the organs in the second whorl are chimeric with petaloid and antheroid tissue. The carpel primordia in whorl 4 are arrested.

(C) Detail of (B) as indicated by the box. Antheroid tissue developing on top of the second whorl organs. Sporogenous tissue with developing pollen is indicated by the arrow.

(D) Female flower of T340-1 with superior carpels in whorl 1 and a malformed ovary in the fourth whorl. In the second whorl, remnants of petal tissue is indicated by an arrow.

(E) Longitudinal section through a female flower of T340-1. Antheroid tissue developing on top of the second whorl organs is indicated in the box. The inner part of the flower is highly malformed and organs are not recognizable.

(F) Detail of (E) as indicated by the box. An arrow shows the developing pollen in the antheroid tissue.

(G) Longitudinal view of a male flower bud of transformant T340-3 in which CUM1 was cosuppressed. Indeterminate floral buds are visible in whorls 3 and 4.

(H) Male flower of transformant T340-3 showing indeterminate floral bud formation in positions normally occupied by stamens. The flower is indeterminate in the center.

(I) Female flower of T340-3, with petal formation in the third whorl. The inferior ovary is not affected in this homeotic mutant.

(J) Detail of (I) showing the petals in whorl 3 and the indeterminacy in the fourth whorl.

(K) Longitudinal view of a female flower bud of T340-3. The small fruit growing inside the primary ovary is indicated by an arrow. The line indicates the plane of the cross-section shown in (L).

(L) Cross-section through a female flower bud of T340-3. The position of the section is indicated in (H). Petals develop in whorl 3 on positions normally occupied by stamens. The whorl 4 structure is the upper part of the small fruit that is growing inside the primary ovary.

The whorl numbers indicate the positions of the floral organs within the flower. S, stigmatic tissue O, ovary. Bars in (B), (E), and (L) = 1 mm.

Interestingly, in these cosuppression flowers, nonreproductive organs were allowed to form in the two inner whorls of male and female flowers at positions where the development of reproductive organs was normally arrested.


Affiliations

Department of Plant Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA

Jong-Kuk Na, Jianping Wang & Ray Ming

Molecular Breeding Division, National Academy of Agricultural Science, RDA, Suwon, 441-701, Republic of Korea

Department of Agronomy, Genetics Institute, Plant Molecular and Cellular Biology program, University of Florida, Gainesville, FL, 32610, USA

FAFU and UIUC-SIB Joint Center for Genomics and Biotechnology, Fujian Agriculture and Forestry University, Fuzhou, Fujian, 350002, China


Climate change’s effect on Rocky Mountain plant is driven by sex

Irvine, Calif., June 30, 2016 — For the valerian plant, higher elevations in the Colorado Rocky Mountains are becoming much more co-ed. And the primary reason appears to be climate change.

In a study appearing July 1in Science, University of California, Irvine environmental biologists Kailen Mooney and Will Petry and colleagues report that an altering climate over the past four decades has significantly changed the growth patterns of male and female Valeriana edulis over elevation. Their work is the first to fully explain sex-specific species responses to climate change.

Valerian is dioecious, meaning individuals are either male or female. Unlike the majority of flowering plants, these cannot self-fertilize. Other well-known dioecious species include asparagus, ginko, papaya, holly, spinach, pistachio, willow and aspen.

In the Colorado Rockies, the sex ratio of valerian populations traditionally changed with climate from low elevation (50 percent male), where it’s hot and dry, to high elevation (only 20 percent male), where it’s cool and wet. At the highest elevations, the rarity of pollen-releasing males reduces the number of seeds produced by female plants.

Now all that’s changing. Over the past 40 years, tests conducted through the Rocky Mountain Biological Laboratory in Crested Butte, Colo., have revealed the region to be warming and drying to such a degree that each valerian population across the elevation gradient is now experiencing a climate that was historically found at a much lower elevation.

Mooney and Petry said their study shows that as the drier, warmer climate moves “up slope,” so do the arid-adapted males, shifting the sex ratios. Because of this, populations in which males were formerly rare now experience less mate limitation, enabling females to successfully produce more seed.

“Nearly all animals and many plants have separate males and females, and they almost always differ in characteristics that affect how they interact with the environment,” said Petry, who earned a Ph.D. in ecology & evolutionary biology at UCI this spring. “Understanding the responses of both sexes is important, because each sex must find mates of the opposite sex to reproduce, and no past work has connected ecological differences between males and females to their responses to climate change and the subsequent consequences for populations.”

These elevation-based patterns of sex ratio change are due, at least in part, to a physiological difference in how males and females use water.

While the increase in males has led to flourishing valerian growth at higher altitudes, an excess of males at low elevations may ultimately result in population declines. In this way, the plants’ sex-specific responses to climate change may cause the species to shift to higher elevations.

Furthermore, fluctuations in the relative abundance of valerian males and females may also have repercussions for species associated with this plant, as the two sexes support different communities of insects.

“Most past work documenting ecological responses to climate change has focused on range shifts of whole species,” said Mooney, an associate professor of ecology & evolutionary biology. “In our study, we instead looked at a species characteristic – the population sex ratio. We’re discovering that males and females respond to climate change differently and that the pace at which this species characteristic responds to climate change is unprecedentedly fast – about 10 times the average rate that species ranges are moving in response to a changing climate.”

Judith Soule, Amy Iler, Ana Chicas-Mosier and David Inouye of the Rocky Mountain Biological Laboratory and Tom Miller of Rice University contributed to the study, which received support from the National Science Foundation (grants DEB-1457029 and DEB-1407318).


Papaya

In some parts of the world, especially Australia and some islands of the West Indies, it is known as papaw, or pawpaw, names which are better limited to the very different, mainly wild Asimina triloba Dunal, belonging to the Annonaceae. While the name papaya is widely recognized, it has been corrupted to kapaya, kepaya, lapaya or tapaya in southern Asia and the East Indies. In French, it is papaye (the fruit) and papayer (the plant), or sometimes figuier des Iles . Spanish-speaking people employ the names melón zapote, lechosa, payaya (fruit), papayo or papayero (the plant), fruta bomba , mamón or mamona , depending on the country. In Brazil, the usual name is mamao . When first encountered by Europeans it was quite naturally nicknamed "tree melon".

Fig. 94: A healthy papaya ( Carica papaya ) in Homestead, Florida, in 1946, when virus diseases were not prevalent.

Commonly and erroneously referred to as a "tree", the plant is properly a large herb growing at the rate of 6 to 10 ft (1.8-3 m) the first year and reaching 20 or even 30 ft (6-9 m) in height, with a hollow green or deep-purple stem becoming 12 to 16 in (30-40 cm) or more thick at the base and roughened by leaf scars. The leaves emerge directly from the upper part of the stem in a spiral on nearly horizontal petioles 1 to 3 1/2 ft (30-105 cm) long, hollow, succulent, green or more or less dark purple. The blade, deeply divided into 5 to 9 main segments, each irregularly subdivided, varies from 1 to 2 ft (30-60 cm) in width and has prominent yellowish ribs and veins. The life of a leaf is 4 to 6 months. Both the stem and leaves contain copious white milky latex.

The 5-petalled flowers are fleshy, waxy and slightly fragrant. Some plants bear only short-stalked pistillate (female) flowers, waxy and ivory-white or hermaprodite (perfect) flowers (having female and male organs), ivory-white with bright-yellow anthers and borne on short stalks while others may bear only staminate (male) flowers, clustered on panicles to 5 or 6 ft (1.5-1.8 m) long. There may even be monoecious plants having both male and female flowers. Some plants at certain seasons produce short-stalked male flowers, at other times perfect flowers. This change of sex may occur temporarily during high temperatures in midsummer. Some "all-male" plants occasionally bear, at the tip of the spray, small flowers with perfect pistils and these produce abnormally slender fruits. Male or hermaphrodite plants may change completely to female plants after being beheaded.

Generally, the fruit is melon-like, oval to nearly round, somewhat pyriform, or elongated club-shaped, 6 to 20 in (15-50 cm) long and 4 to 8 in (10-20 cm) thick weighing up to 20 lbs (9 kg). Semi-wild (naturalized) plants bear miniature fruits 1 to 6 in (2.5-15 cm) long. The skin is waxy and thin but fairly tough. When the fruit is green and hard it is rich in white latex. As it ripens, it becomes light- or deep-yellow externally and the thick wall of succulent flesh becomes aromatic, yellow, orange or various shades of salmon or red. It is then juicy, sweetish and somewhat like a cantaloupe in flavor in some types quite musky. Attached lightly to the wall by soft, white, fibrous tissue, are usually numerous small, black, ovoid, corrugated, peppery seeds about 3/16 in (5 mm) long, each coated with a transparent, gelatinous aril.

Though the exact area of origin is unknown, the papaya is believed native to tropical America, perhaps in southern Mexico and neighboring Central America. It is recorded that seeds were taken to Panama and then the Dominican Republic before 1525 and cultivation spread to warm elevations throughout South and Central America, southern Mexico, the West Indies and Bahamas, and to Bermuda in 1616. Spaniards carried seeds to the Philippines about 1550 and the papaya traveled from there to Malacca and India. Seeds were sent from India to Naples in 1626. Now the papaya is familiar in nearly all tropical regions of the Old World and the Pacific Islands and has become naturalized in many areas. Seeds were probably brought to Florida from the Bahamas. Up to about 1959, the papaya was commonly grown in southern and central Florida in home gardens and on a small commercial scale. Thereafter, natural enemies seriously reduced the plantings. There was a similar decline in Puerto Rico about 10 years prior to the setback of the industry in Florida. While isolated plants and a few commercial plots may be fruitful and long-lived, plants in some fields may reach 5 or 6 ft, yield one picking of undersized and misshapen fruits and then are so affected by virus and other diseases that they must be destroyed.

In the 1950's an Italian entrepreneur, Albert Santo, imported papayas into Miami by air from Santa Marta, Colombia, Puerto Rico and Cuba for sale locally as well as shipping fresh to New York, and he also processed quantities into juice or preserves in his own Miami factory.

Since there is no longer such importation, there is a severe shortage of papayas in Florida. The influx of Latin American residents has increased the demand and new growers are trying to fill it with relatively virus-resistant strains selected by the University of Florida Agricultural Research and Education Center in Homestead.

Successful commercial production today is primarily in Hawaii, tropical Africa, the Philippines, India, Ceylon, Malaya and Australia, apart from the widespread but smaller scale production in South Africa, and Latin America.

Annual papaya consumption in Hawaii is 15 lbs (6.8 kg) per capita, yet 26 million lbs (11,838,700 kg) of fresh fruits were shipped by air freight to mainland USA in 1974, mainly direct from Hilo or via Honolulu.

Puerto Rican production does not meet the local demand and fruits are imported from the Dominican Republic for processing.

The papaya is one of the leading fruits of southern Mexico and 40% of that country's crop is produced in the state of Veracruz on 14,800 acres (6,000 ha) yielding 120,000 tons annually.

Fruits from bisexual plants are usually cylindrical or pyriform with small seed cavity and thick wall of firm flesh which stands handling and shipping well. In contrast, fruits from female flowers are nearly round or oval and thin-walled. In some areas, bisexual types are in greatest demand. In South Africa, round or oval papayas are preferred.

Fig. 95: Papaya fruits vary in form, size, thickness, color and flavor of flesh. Favored types have little, if any, muskiness of odor.

Despite the great variability in size, quality and other characteristics of the papaya, there were few prominent, selected and named cultivars before the introduction into Hawaii of the dioecious, small-fruited papaya from Barbados in 1911. It was named 'Solo' in 1919 and by 1936 was the only commercial papaya in the islands. 'Solo' produces no male plants just female (with round, shallowly furrowed fruits) and bisexual (with pear-shaped fruits) in equal proportions. The fruits weigh 1.1 to 2.2 lbs (1/2-1 kg) and are of excellent quality. When the fruit is fully ripe the thin skin is orange-yellow and the flesh golden-orange and very sweet.

'Kapoho Solo' or 'Puna Solo' was discovered and became popular with growers on Kauai before 1950. In 1955 a 'Dwarf Solo' (a back-cross of Florida's 'Betty' and 'Solo') was introduced to aid harvesting, and this became the leading commercial papaya on the island of Oahu. It was, up to 1974, the only export cultivar. It is pear-shaped, 14 to 28 oz (400-800 g) in weight in high rainfall areas, and has yellow skin and pale-orange flesh.

'Waimanalo' (formerly 'Solo' Line 77) was selected in 1960 and released by the Hawaii Agricultural Experiment Station in 1968 and soon superseded Line 8 'Solo' on Oahu for the fresh fruit market because of its firmness and quality, but there it is usually too large for export. It has long storage life and is recommended for sale fresh and for processing. Since 1974 this cultivar has been produced commercially on the low-rainfall island of Maui where it ripens at a greener color than on the island of Hawaii and is exported to cities in the northwestern and central USA. The growers raised only bisexual plants they say that the fruits of female plants are too rough in appearance.

'Higgins' (formerly Line 17A), the result of crosses in 1960, was introduced to Hawaiian growers in 1974. It is of high quality, pear-shaped, with orange-yellow skin, deep-yellow flesh, and averages 1 lb (0.45 kg) when grown under irrigation. In and territory or seasons of low rainfall, the fruit is undersized.

'Wilder' (formerly Line 25) is a cultivar admired for its uniformity of size, firmness and small cavity and it is now popular for export.

'Hortus Gold', a South African cultivar, launched in the early 1950's, is dioecious, early-maturing, with round-oval, golden-yellow fruits, 2 to 3 lbs (0.9-1.36 kg) in weight. From 200 female 'Hortus Gold' seedlings planted at the University of Natal's Ukulinga Research Farm in 1960, selections were made of the plants showing the highest yield. Of these, one clone having the best sugar content and disease resistance was chosen and named 'Honey Gold' in 1976. This cultivar has a slight beak at the apex, golden-yellow skin is of sweet flavor and good texture but becomes mushy when overripe. It averages 2.2 lbs (1 kg) per fruit except for those at the end of the season which are much smaller. It does not reproduce true from seed and is therefore propagated by cuttings. It is late in season and late-maturing (10 months from fruit set to maturity) and therefore brings nearly double the price of other cultivars.

'Bettina' and 'Petersen' , long-standing cultivars in Queensland, Australia, were inbred for several generations to obtain pure lines. 'Bettina', a hybrid of Florida's 'Betty' and a Queensland strain, is a low, shrubby, dioecious plant producing well-colored, round-oval fruits weighing 3 to 5 lbs (1.36-2.27 kg).

'Improved Petersen' , of local origin, is dioecious, tall-growing, with fruits deficient in external color and indifferent as to keeping quality but noted for the fine color and flavor of the flesh. In 1947 'Bettina 100A' was crossed with 'Petersen 170' to produce the superior, semi-dwarf 'Hybrid No. 5' , smooth, yellow, rounded-oval, 3 lbs (1.36 kg) in weight, thick-fleshed, of excellent flavor and prized for marketing fresh and for canning. It bore more heavily than either of its parents and remained a preferred cultivar for more than 20 years. 'Solo' and 'Hortus Gold' are often grown but most plantations are open-pollinated mixtures.

In Western Australia, after trials of 9 cultivars –'Hybrid No. 5', 'Petersen', 'Yarwun Yellow', 'Gold Cross', 'Goldy', 'Hong Kong', 'Guinea Gold', 'Golden Surprise' and 'Sunnybank'– only 'Sunnybank' and 'Guinea Gold' were chosen as having sufficient yield and quality to be worth cultivating commercially. 'Sunnybank' fruits average 1.39 lbs (0.63 kg), and ripen over 11 months. 'Guinea Gold' averages 2.4 lbs (a little over 1 kg) and ripens over a period of 18 months.

The Universidad Agraria, La Molina, Peru, began to assemble papaya strains in 1964, collecting 40 from various parts of the country and introducing 3 from Brazil, 1 from Puerto Rico, 3 from Mexico and 2 lines of 'Solo' from Hawaii, and embarked on an evaluation and breeding program and the creation of a germplasm bank.

In Ghana, dioecious cultivars such as 'Solo', 'Golden Surprise', 'Hawaii', and 'No. 5595', were introduced and commonly cultivated by farmers but they hybridized with local types and lost their identities after several generations. A number of types were collected at the Agricultural Research Station at Kade from 1966 to 1970 and classified according to sex type, fruit form, weight, skin and flesh color, flesh thickness, texture and flavor, number of seeds, and various plant factors. It was determined that preference should be given female plants with short, stout stems, early maturing, and bearing heavily all year medium-size fruits of bright color, thick-flesh and with few seeds.

The Instituto Colombiano Agropecuario, at Palmira, Colombia, began a papaya breeding program in 1963 by bringing together Colombian-grown cultivars –'Campo Grande', 'Tocaimera', 'Zapote', 'Solo', –with some from Brazil –'Betty', 'Bettina' and '43-A-3' –South Africa– 'Hortus Gold'– and Puerto Rico, and representatives of related species: C. candamarcensis Hook. F., C. pentagona Heilborn, C. goudotiana Tr. & Pl. (one type yellow with green peduncles and another red with purple peduncles), C. cauliflora Jacq. of Colombia and C. monoica Desf. and Jacaratia dodecaphylla A. DC. from Peru.

The first two of these species were not suited to conditions at Palmira.

The progeny of crosses with C. caulfliora were the only hybrids showing some virus resistance but they were unfruitful when attacked. There were no viable seeds and 30% of the fruits were seedless. C. monoica proved well adapted to Palmira, bore small, yellow fruits, but succumbed to virus. The introductions from Brazil were by far the most promising. 'Zapote' , with rich, red flesh is much grown on the Atlantic coast of Colombia.

In India, papaya breeding and selection work has been carried on for over 30 years beginning with 100 introduced strains and 16 local variations. A well-known cultivar is 'Coorg Honey Dew', a selection from 'Honey Dew' at Chethalli Station of the Indian Institute of Horticultural Research. There are no male plants female and bisexual occur in equal proportions. The plant is low-bearing and prolific. The fruit is long to oval, weighs 4.4 to 7.7 lbs (2-3 1/2 kg) has yellow flesh with a large cavity, and keeps fairly well. 'Washington', popular in Bombay, has dark-red petioles and yellow flowers. The fruits are of medium size with excellent, sweet flavor. 'Burliar Long' is prolific, bearing as many as 103 fruits the first year, mostly in pairs densely packed along the stem down to 18 in (45 cm) from the ground. Seedlings are 70% females and bloom 3 months after transplanting.

'Co. 1' and 'Co. 2' were developed at Tamil Nadu Agricultural University. Both are dioecious and dwarf, the first fruits being borne 3 ft (1 m) from the ground. 'Co. 1' is valued for eating fresh 'Co. 2' is grown for table use and for papain extraction. The fruits are of medium sizeן.3 to 5.5 lbs (1 1/2-2 1/2 kg), with yellow, sweet flesh.

The Regional Research Station at Pusa has introduced some promising selections:

'Pusa Delkious' ('Pusa 1-15')–medium size flesh deep-orange, of excellent flavor female and hermaphrodite plants high-yielding.

'Pusa Majesty' ('Pusa 22-3')–round, of medium size flesh yellowish, solid keeps well and ships well vinis resistant hermaphrodite plants higher-yielding than the female.

'Pusa Giant' ('Pusa 1-45V')–large fruits suitable for marketing ripe, or green for use as a vegetable, also for canning. Plant dioecious, fast-growing tall trunk thick, wind-resistant.

'Pusa Dwarf' ('Pusa 1-45')–fruit oval, of medium size. Plant is dwarf begins bearing fruit at 10 to 12 in (25-30 cm) above the ground. In much demand for home and commercial culture suitable for high-density plantings.

In 1965, a program of papaya improvement was undertaken in Trinidad and Tobago utilizing promising selections from local types, including 'Santa Cruz Grant', a vigorous plant mainly bisexual (having both male and female flowers), very large fruits weighing 10 to 15 lbs (4.5-6.8 kg), with firm, yellow flesh of agreeable flavor. The fruit is too large for marketing fresh but is processed both green and ripe. 'Cedro' is dioecious, rarely bisexual, a heavy bearer and highly resistant to anthracnose. The fruits weigh from 3 to 8 lbs (1.37-3.6 kg) but average 6 lbs (2.7 kg) have firm, yellow, melon-like flesh and are suitable for sale fresh or for processing.

In 'Singapore Pink', the plants are mainly bisexual, producing cylindrical fruit. The minority are female with round fruit. Average weight of fruit is 5 lbs (2.27 kg) though there is variation from 2 to 7 lbs (1-3 kg). The flesh is pink. The fruit surface is prone to anthracnose in rainy periods, so, at such times, the fruits must be picked and sold in the green state. Two smaller-fruited types, 2 to 3 lbs (1-1.37 kg) in weight, with bright-yellow skin and thick, firm flesh, were selected for marketing fresh.

The 'Solo' of Hawaii has performed unsatisfactorily in Florida, producing low yields of small fruits. Scott Stambaugh, a papaya specialist, began his papaya breeding with a strain designated USDA Bureau of Plant Industry #28533 obtained from the then Plant Introduction Station in Miami. From offspring of this he made a selection which he named 'Norton'. When he acquired seed of a type called 'Purplestem' later 'Bluestem', he crossed it with 'Norton' and the hybrid yielded fruits 10 lbs (4.5 kg) in weight and was named 'Big Bluestem'. The latter was crossed with 'Solo' and the hybrid was called 'Bluestem Solo' or 'Blue Solo'. The 'Blue Solo' has been well regarded in Florida for its low growth, dependable yields of good quality fruits, 2 to 4 lbs (1-2 kg) in weight, orange-fleshed and rich in flavor.

'Cariflora' is a new cultivar developed at the recently renamed Tropical Research and Education Center of the University of Florida at Homestead. It is nearly round, about the size of a cantaloupe, with thick, dark-yellow to light-orange flesh tolerant of papaya ringspot virus, but not resistant to papaya mosaic virus or papaya apical necrosis virus. Yield is good in southern Florida and warm lowlands of tropical America but not at elevations above 2625 ft (800 m).

'Sunrise Solo' (formerly HAES 63-22) was introduced from Hawaii into Puerto Rico. The fruit has pink flesh with high total solid content. In Puerto Rican trials, seeds were planted in mid-November, seedlings were transplanted to the field 2 months later, flowering occurred in April and mature fruits were harvested from early August to January. Recent selections from Puerto Rican breeding programs are 'P.R. 6-65' (early), 'P.R. 7-65' (late), and 'P.R. 8-65'.

Venezuelan papayas are usually long and large, ranging in weight from 2 to 13 lbs (1-6 kg) and mostly for domestic consumption or shipment by boat to nearby islands.

If a papaya plant is inadequately pollinated, it will bear a light crop of fruits lacking uniformity in size and shape. Therefore, hand-pollination is advisable in commercial plantations that are not entirely bisexual.

Bags are tied over bisexual blossoms for several days to assure that they are self-pollinated. The progeny of self-pollinated bisexual flowers are 67% bisexual, the rest being female.

To cross-pollinate, one or 2 stamens from a bisexual flower are placed on the pistil of a female flower about to open and a bag is tied over the flower for a few days. Most of such cross-pollinated blooms should set fruit. Resulting seeds will produce 1/2 female and 1/2 bisexual plants.

By another method, all but the apical female flower bud are removed from a stalk and the apical bud is bagged 1-2 days before opening. At full opening, the stigma is dusted with pollen from a selected male bloom and the bag quickly resealed and it remains so for 7 days.

Plants from female flowers crossed with male flowers are 50-50 male and female. Bisexual flowers pollinated by males give rise to 1/3 female, 1/3 bisexual and 1/3 male plants.

South African growers have long been urged to practice hand-pollination in order to maintain a selected strain and, in breeding, to incorporate factors such as purple stem, yellow flowers and reddish flesh so that the improved selection will be distinguishable from ordinary strains with non-purple stems, white flowers and yellow flesh.

The papaya is a tropical and near-tropical species, very sensitive to frost and limited to the region between 32º north and 32º south of the Equator. It needs plentiful rainfall or irrigation but must have good drainage. Flooding for 48 hours is fatal. Brief exposure to 32º F (-0.56º C) is damaging prolonged cold without overhead sprinkling will kill the plants.

While doing best in light, porous soils rich in organic matter, the plant will grow in scarified limestone, marl, or various other soils if it is given adequate care. Optimum pH ranges from 5.5 to 6.7. Overly acid soils are corrected by working in lime at the rate of 1-2 tons/acre (2.4-4.8 tons/ha). On rich organic soils the papaya makes lush growth and bears heavily but the fruits are of low quality.

Papayas are generally grown from seed. Germination may take 3 to 5 weeks. It is expedited to 2 to 3 weeks and percentage of germination increased by washing off the aril. Then the seeds need to be dried and dusted with fungicide to avoid damping-off, a common cause of loss of seedlings. Well-prepared seeds can be stored for as long as 3 years but the percentage of germination declines with age. Dipping for 15 seconds in hot water at 158º F (70º C) and then soaking for 24 hrs in distilled water after removal from storage will improve the germination rate. If germination is slow at some seasons, treatment with gibberellic acid may be needed to get quicker results.

To reproduce the characteristics of a preferred strain, air-layering has been successfully practiced on a small scale. All offshoots except the lowest one are girdled and layered after the parent plant has produced the first crop of fruit. Later, when the parent has grown too tall for convenient harvesting the top is cut off and new buds in the crown are pricked off until offshoots from the trunk appear and develop over a period of 4 to 6 weeks. These are layered and removed and the trunk cut off above the originally retained lowest sprout which is then allowed to grow as the main stem. Thereafter the layering of offshoots may be continued until the plant is exhausted.

Rooting of cuttings has been practiced in South Africa, especially to eliminate variability in certain clones so that their performance can be more accurately compared in evaluation studies. Softwood cuttings made in midsummer rooted quickly and fruited well the following summer. Cuttings taken in fall and spring were slow to root and deficient in root formation. The commercial cultivar 'Honey Gold' is grown entirely from cuttings. Once rooted, the cuttings are planted in plastic bags and kept under mist for 10 days, and then put in a shade house for hardening before setting in the field.

Hawaiian workers have found that large branches 2-3 ft (60-90 cm) long rooted more readily than small cuttings. Planted 1 ft (30 cm) deep in the rainy season, they began fruiting in a few months very close to the ground.

In budding experiments both Forkert and chip methods have proved satisfactory in Trinidad. However, it is reported that a vegetatively propagated selected strain deteriorates steadily and is worthless after 3 or 4 generations.

In Hawaii, 'Solo' grafted onto 'Dwarf Solo' was reduced in vigor and productivity, but 'Dwarf Solo' grafted onto 'Solo' showed improved performance.

In recent years, the potential of rapid propagation of papaya selections by tissue culture is being explored and promises to be feasible even for the establishment of commercial plantations of superior strains.

Efforts have been made to determine the sex of seedlings in the nursery, Indian scientists making colorimetric tests of leaf extracts have had 87% success in identifying seedlings as female 67% in classifying males/bisexuals grouped together.

Planting may be done at any time of year and local conditions determine when it is best for the crop to come in. Papayas mature in 6 to 9 months from seed in the hotter areas of South Africa in 9 to 11 months where it is cooler, providing an opportunity to supply markets in the off-season when prices are high. Seeds planted in early summer or midsummer will produce the first crop in the second winter. Thereafter, the same plants will mature fruit from spring to early summer. Spring fruits are apt to be sunburned because of winter leaf loss are also subject to fruit spot and have a low sugar content. Sunburn can be avoided by advance whitewashing of sides exposed to the afternoon sun. Some growers manipulate the harvest season by stripping off 6 of the newly set fruits, thus forcing the plant to bloom again and produce fruits 6 to 8 weeks later than they normally would.

In southern Florida, plants set out in March or April will ripen their fruits in November and December and have the advantage of a "tourist" market. July plantings will be slowed down by winter and will not fruit for 10 months or more. Some growers advocate planting in September and October so that the crop will be ready for harvest before the onset of the main hurricane season. Further north in the state, papayas must be set out in March or April in order to have the required growing season before frost.

Puerto Rican trials have shown that papaya plants set in the field on 6 ft (1.8 m) centers made stronger, stouter growth and were more fruitful than those at closer spacings. Some growers insist on an 8 x 8 ft (2.4 x 2.4 m) area per plant. In India, 'Co. 1' and 'Co. 2' and 'Solo' are set on 6 ft (1.8 m) centers 'Coorg Honey Dew' and 'Washington' on 8 ft (2.4 m) centers. Princess Orchards on Maui, Hawaii, plant in double rows with an alley between each pair providing room for cultural and harvesting operations. In Queensland, plants may be set only 3 ft (1 m) apart on level ground and then thinned out by removal of unwanted plants after flowering.

Seeds may be planted directly in the field, or seedlings raised in beds or pots may be transplanted when 6 weeks old or even up to 6 months of age, though there must be great care in handling and the longer the delay the greater the risk of dehydrated or twisted roots also, transplanting often results in trunk-curvature in windy locations.

Experiments in Hawaii indicate that direct seeding results in deeper tap-roots, erect and more vigorous growth, earlier flowering and larger yields.

In Puerto Rico, it is customary to set 2 plants per hole. In El Salvador planters place 5 to 6 seeds, separated from each other, in each hole at a depth of 3/8 in (1 cm). When the plants bloom, 90% of the males are removed, preferably by cutting off at ground level. Pulling up disturbs the roots of the remaining plants. If the plantation is isolated and there is no chance of cross-pollination by males, all the seed will become female or hermaphrodite plants. Fruits should mature 5 to 8 months later.

In India, seeds are usually treated with fungicide and planted in beds 6 in (15 cm) above ground level that have been organically enriched and fumigated. The seeds are sown 2 in (5 cm) apart and 3/4 to 1 1/8 in (2-3 cm) deep in rows 6 in (15 cm) apart. They are watered daily and transplanted in 2 1/2 months when 6 to 8 in (15-20 cm) high. Transplanting is more successful if polyethylene bags of enriched soil are used instead of raised beds. Two seeds are planted in each bag but only the stronger seedling is maintained. Transplanting is best done in the evening or on cloudy, damp days. On hot, dry days, each plant must be protected with a leafy branch or palm leaf stuck in the soil. Except for 'Coorg Honey Dew' and 'Solo', the plants are set out in 3's, 6 in (15 cm) apart in enriched pits. After flowering, one female or hermaphrodite plant is retained, the other two removed. But one male is kept for every 10 females. 'Coorg Honey Dew' and 'Solo' are planted one to a pit and no males are necessary. Watering is done every day until the plants are well established, but overwatering is detrimental to young plants. Double rows of Sesbania aegyptiaca are planted as a windbreak.

The installation of constant drip irrigation (12 gals per day) has made possible papaya cultivation on mountain slopes on the relatively dry island of Maui which averages 10 in (25 cm) of rain annually.

Papaya plants require frequent fertilization for satisfactory production. In India, best results have been obtained by giving 9 oz (250 g) of nitrogen, 9 oz (250 g) of phosphorus, and 18 oz (500 g) potash to each plant each year, divided into 6 applications.

Because of the need to expedite growth and production before the onslaught of diseases, Puerto Rican agronomists recommend treating the predominantly clay soil with a nematicide before planting, giving each plant 4 oz (113 g) of 15-15-15 fertilizer at the end of the first week, and each month thereafter increasing the dose by 1 oz (28 g) until the beginning of flowering, then applying .227 g per plant as a final treatment. In trials, this program has permitted 6 harvests of green fruits for processing, each over 1 lb (1/2 kg) in weight, spanning a period of 13 months. The roots usually extend out beyond the leaves and it is advisable to spread fertilizer over the entire root area.

In late fertilizer applications of a crop destined for canning, nitrogen should be omitted because it renders the fruit undesirable for processing. High nitrate content in canned papaya (as with several common vegetables) removes the tin from the can. To avoid nitrogen deficiency at the beginning of flowering for the next crop, 1 or 2% urea sprays can be applied.

In southern Florida, on oolitic limestone, experts have prescribed liquid fertilizer weekly for the first 10 weeks and then 1 lb (1/2 kg) of 4-8-6 dry fertilizer mixture (with added minor elements) per plant weekly until flowering. Here a heavy organic mulch is desirable to conserve moisture, control weeds, keep the soil cool, and help repel nematodes.

Mechanical cultivation between rows is apt to disturb the shallow roots. judicious use of herbicides is preferable.

Overcrowded fruits should be thinned out when young to provide room for good form development and avoid pressure injury. Cold weather may interfere with pollination and cause shedding of unfertilized female flowers. Spraying the inflorescence with growth regulators stops flower drop and significantly enhances fruit set. After the first crop, the terminal growth may be nipped off to induce branching which tends to dwarf the plant and facilitates harvesting. However, unless the plants are strong growers, fruiting branches may need to be propped to avoid collapse.

Studies in Hawaii have shown that papaya flavor is at its peak when the skin is 80% colored. For the local market, in winter months, papayas may be allowed to color fairly well before picking, but for local market in summer and for shipment, only the first indication of yellow is permissible. The fruits must be handled with great care to avoid scratching and leaking of latex which stains the fruit skin. Home growers may twist the fruit to break the stem, but in commercial operations it is preferable to use a sharp knife to cut the stem and then trim it level with the base of the fruit. However, to expedite harvesting of high fruits, most Hawaiian growers furnish their pickers with a bamboo pole with a rubber suction cup (from the well-known "plumber's helper") at the tip. With the cup held against the lower end of the fruit, the pole is thrust upward to snap the stem and the falling fruit is caught by hand. One man can thus gather 800-1,000 lbs (363-454 kg) daily.

In Hawaii, it has been calculated that manual picking and field sorting constitute 40% of the labor cost of the crop (1,702 man-hours per acre to pick and pack). Therefore, in 1970, an experimental mechanical aid was tested and results indicated that a machine with one operator and 2 pickers could harvest 1,000 lbs (454 kg) of fruit per hour, the equivalent of 8 men hand-picking. Many factors, such as investment, operation and repair costs, useful life, and so forth must be considered before such a machine could be determined to be feasible. On the island of Maui, harvesting is aided by hydraulic lifts, each operated by a single worker. Picking starts when the plants are 11 months of age and continues for 48 months when the trees are 25 ft (7.5 m) high, too tall for further usefulness.

The fruits are best packed in single layers and padded to avoid bruising. The latex oozing from the stem may irritate the skin and workers should be required to wear gloves and protective clothing.

In the usual papaya plantation, each plant may ripen 2 to 4 fruits per week over the fruiting season. Healthy plants, if well cared for, may average 75 lbs (34 kg) of fruit per plant per year, though individual plants have borne as much as 300 lbs (136 kg). In South Africa, branched 'Honey Gold' plants set 20 ft (6 m) apart in rows 10 ft (3 m) apart have produced 45 lbs (100 kg) of fruit each in their 4th year. A field of 1,000 plants occupying 2 1/2 acres (1 ha) gave 30 tons of fruit. In the Hilo area of the island of Hawaii, production averages 15 tons per acre (37 tons/ha). From 250 acres (100 ha), Princess Orchards on Maui harvests 150,000 lbs (68,180 kg) weekly during the season.

In the Kapoho region of the island of Hawaii, yields average 38,000 lbs/acre (roughly 38,000 kg/ha) the first year, 25,000 lbs (11,339 kg) the second year. Papaya plants bear well for 2 years and then productivity declines and commercial plantings are generally replaced after 3-4 years. By that time they have attained heights which make harvesting difficult.

In Trinidad and Tobago, plants that have become too tall are cut to the ground and side shoots are allowed to grow and bear. In El Salvador, after the 3rd year of bearing, the main stem is cut off about 3 ft (1 m) from the ground at the beginning of winter and is covered with a plastic bag to protect it from rain and subsequent rotting. Several side shoots will emerge within a few days. When these reach 8 in to 1 ft (20-30 cm) in height, all are cut off except the most vigorous one which replaces the original top.

Fruits can be held at 85º F (29.64º C) and high atmospheric humidity for 48 hours to enhance coloring before packing. Standard decay control has been a 20-minute submersion in water at 120º F (49º C) followed by a cool rinse. In India, dipping in 1,000 ppm of aureofungin has been shown to be effective in controlling postharvest rots. In Philippine trials, thiabendazole reduced fruit rot by 50%. In 1979, Hawaiian workers demonstrated that spreading an aqueous solution of carnauba wax and thiabendazole over harvested fruits gives good protection from postharvest diseases and can eliminate the hot-water bath.

In Puerto Rico, fruits of 'P.R. 8-65', picked green, were ripened successfully by 6-7 days treatment with ethylene gas in airtight chambers at 77º F (25º C) and 85 to 95% humidity, following the hot-water bath.

Hawaiian papayas must be sanitized before shipment to the mainland USA to avoid introduction of fruit flies. Fruits picked 1/4 ripe are prewarmed in water at 110º F (43.33º C) for about 40 min, then quickly immersed for 20 min at 119º (48.33º C). This double-dipping maybe replaced by irradiation. One little-used method is a vapor-heat treatment following dry heat at 110º F (43.33º C) and 40% relative humidity.

Fruits that have had hot water treatment and EDB fumigation and then have been stored in 1.5% oxygen at 55º F (13º C) for 12 days will have a shelf life of about 3 1/2 days at room temperature. Fruits that have had hot water treatment when 1/4 colored, followed by irradiation at 75-100 krad, and storage at 2-4% oxygen and 60º F (16º C) for 6 days will have a market life of 8 days. Those held for 12 days will be saleable thereafter for 5 days.

In Puerto Rico, gamma irradiation (25-50 krads) delayed ripening up to 7 days. Treatment at 100 krads slightly accelerated ripening in storage. Even at the lowest level irradiation inhibited fungal growth. Carotenoid content was unaffected but ascorbic acid was slightly reduced at all exposures.

Partly ripe papayas stored below 50º F (10º C) will never fully ripen. This is the lowest temperature at which ripe papayas can be held without chilling injury.

'Solo 62/3' fruits harvested in Trinidad at the first sign of yellow, treated with fungicide, placed in perforated polyethylene bags and packed in individual compartments in cartons, have been shipped to England by air (2 days' flight), ripened at 68º F (20º C), and found to be of excellent quality and flavor.

The same cultivar, similarly handled, withstood transport in the refrigerated hold of a ship for 21 days. Immediately ripened on arrival, the fruits were well accepted on the London market. Sea shipment proved to be the more economical.

Hypobaric (low pressure) containers have made possible satisfactory sea shipment (18-21 days) of hot-water treated and fungicidal-waxed papayas from Hilo, Hawaii, to Los Angeles and New York.

A major hazard to papayas in Florida and Venezuela is the wasp-like papaya fruit fly, Toxotrypana curvicauda. The female deposits eggs in the fruit which will later be found infested with the larvae. Only thick-fleshed fruits are safe from this enemy. Control on a commercial scale is very difficult. Home gardeners often protect the fruit from attack by covering with paper bags, but this must be done early, soon after the flower parts have fallen, and the bags must be replaced every 10 days or 2 weeks as the fruits develop. Rolled newspaper may be utilized instead of bags and is more economical. India has no fruit fly with ovipositor long enough to lay eggs inside papayas.

An important and widespread pest is the papaya web-worm, or fruit cluster worm, Homolapalpia dalera, harbored between the main stem and the fruit and also between the fruits. It eats into the fruit and the stem and makes way for the entrance of anthracnose. Damage can be prevented if spraying is begun at the beginning of fruit set, or at least at the first sign of webs.

The tiny papaya whitefly, Trialeuroides variabilis, is a sucking insect and it coats the leaves with honeydew which forms the basis for sooty mold development. Shaking young leaves will often reveal the presence of whiteflies. Spraying or dusting should begin when many adults are noticed. Hornworms (immature state of the sphinx moth– Erinnyis obscura in Jamaica, E. ello in Venezuela, E. alope in Florida) feed on the leaves, as do the small, light-green leafhoppers.

Mention is made later on of the aphids that transmit virus diseases and other infections.

Other pests requiring control measures in Australia include the red spider, or red spider mite, Tetranychus seximaculatus, which sucks the juice from the leaves. In India and on the island of Maui, plant and fruit infestation by red spider has been a major problem. This pest and the cucumber fly and fruit-spotting bugs feed on the very young fruits and cause them to drop. In Hawaii, the red-and-black-flat mite feeds on the stem and leaves and scars the fruit. The broad mite damages young plants especially during cool weather.

In the Virgin Islands scale has been most troublesome, apart from rats and fruit-bats that attack ripe fruits. In Australia, 5 species of scale insects have been found on papayas, the most serious being oriental scale, Aonidiella orientalis, which occurs on both the fruit and the stem. So far, it is confined to limited areas. In Florida, the scale insects Aspidiotus destructor and Coccus hesperidium may infest bagged fruit more than unbagged fruit. Another scale, Philaphedra sp., has recently been reported here.

Indian scientists have observed that immature earthworms, Megascolex insignis, are attracted by and feed on rotting tissue of papaya plants. They hasten the demise of plants afflicted with stem rot from Pythium aphanidermatum and may act as vectors for this fungus.

Root-knot nematodes, Meloidogyne incognita acrita, and reniforin nematodes, Rotylenchulus reniformis, are detrimental to the growth and productivity of papaya plants and should be combatted by pre-planting soil fumigation if the nematode population is high.

Hawaii, partly because of its distance from other papaya-growing areas, is less afflicted with disease problems than Florida and Puerto Rico, but still has to combat a number of major and minor maladies. Most serious of all is the mosaic virus, on plant and fruit, which is common in Florida, Cuba, Puerto Rico, Trinidad, and first seen in Hawaii in 1959. It is transmitted mechanically or by the green peach aphid, Myzus Persicae, and other aphids including the green citrus aphid, Aphis spiraecola, in Puerto Rico. Two forms of mosaic virus are reported in Puerto Rico: the long-known "southern coast papaya mosaic virus", the symptoms of which include extreme leaf deformation, and the relatively recent "Isabela mosaic virus" on the northern coast which is similar but without leaf distortion. Both forms occur in some northcoast plantations. There is no remedy, but measures to avoid spread include the destruction of affected plants, control of aphids by pesticides, and elimination of all members of the Cucurbitaceae from the vicinity. Mosaic is sporadic and scattered and not of great concern in Queensland.

Papaya ringspot virus, prevalent in Florida, the Dominican Republic and Venezuela, is occasionally serious in the Waianae area on the dry leeward side of Oahu. It is transmitted by the same vectors. Mosaic and ringspot viruses are the main limiting factors in papaya production in the Cauca Valley of Colombia.

In Florida, virus diseases were recognized as the greatest threat to the papaya industry in the early 1950's. The first signs are irregular mottling of young leaves, then yellowing with transparent areas, leaf distortion, and rings on the fruit. If affected plants are not removed, the condition spreads throughout the plantation. Fruits borne 2 or 3 months after the first symptoms will have a disagreeable, bitter flavor.

At the Agricultural Research and Education Center of the University of Florida in Homestead, the late Dr. Robert Conover established a test plot of papayas grown from seed of 95 accessions from a number of countries and 94 collections in Florida in the hope of finding some virus-free strains. Most of the introductions were highly susceptible to papaya ringspot virus local strains showed some resistance. Highest tolerance was shown by a dioecious, round-fruited, yellow-fleshed strain brought from Colombia by Dr. S.E. Malo several years ago. The fruits weigh 3-5 lbs (1.36-2.27 kg).

It is thought that at least 3 virus diseases are involved in papaya decline in East Africa and it has been suggested that the diseases are spread in part by the tapping of green fruits for their latex (the source of papain).

Bunchy top is a common, controllable mycoplasma disease transmitted by a leafhopper, Empoasca papayae in Puerto Rico, the Dominican Republic, Haiti, and Jamaica by that species and E. dilitara in Cuba and by E. stevensi in Trinidad. Bunchy top can be distinguished from boron deficiency by the fact that the tops of affected plants do not ooze latex when pricked.

In the subtropical part of Queensland, but not in the tropical, wet climate of northern Queensland, papaya plants are subject to die-back, a malady of unknown origin, which begins with shortening of the petioles and bunching of inner crown leaves. Then the larger crown leaves quickly turn yellow. Affected plants can be cut back at the first sign of the disease and if the cut stem is covered to avoid rotting, the top will be replaced by healthy side branches. The problem occurs mainly in the hot, dry spring after a season of heavy rains.

Anthracnose, which usually attacks the ripe fruits and is caused by the fungus Colletotrichum gloeosporioides , was formerly the most important papaya disease in Hawaii, Mexico and India, but it is controllable by spraying every 10 days, or every week in hot, humid seasons, and hotwater treatment of harvested fruits. A strain of this fungus produces "chocolate spot" (small, angular, superficial lesions). A disease resembling anthracnose but which attacks papayas just beginning to ripen, was reported from the Philippines in 1974 and the causal agent was identified as Fusarium solani .

A major disease in wet weather is phytophthora blight. Phytophthora parasitica attacks and rots the stem and roots of the plant and infects and spoils the fruit surface and the stem-end, inducing fruit fall and mummification. Fungicidal sprays and removal of diseased plants and fruits will reduce the incidence. P. Palmivora has been identified as the chief cause of root-rot in Hawaii and Costa Rica. In Hawaii, the strains, 'Waimanalo-23' and -24, 'Line 8' and 'Line 40', are resistant to this fungus. 'Kapoho Solo' and '45-T 22 ' are moderately resistant, and 'Higgins' is susceptible.

Root-rot by Pythium sp. is very damaging to papayas in Africa and India. P. ultimum causes trunk rot in Queensland. Collar rot in 8- to 10-month old seedlings, evidenced by stunting, leaf-yellowing and shedding, and total loss of roots, was first observed in Hawaii in 1970, and was attributed to attack by Calonectria sp. Collar rot is sometimes so severe in India as to cause growers to abandon their plantations.

Powdery mildew, caused by Oidium caricae (the imperfect state of Erysiphe cruciferarum the source of mildew in the Cruciferae) often affects papaya plants in Hawaii and both plants and fruits elsewhere. Sulfur, judiciously applied, is an effective control. Powdery mildew is caused by Sphaerotheca humili in Queensland and by Ovulariopsis papayae in East Africa. Angular leaf spot, a form of powdery mildew, is linked in Queensland to the fungus Oidiopsis taurica .

Corynespora leaf spot, or brown leaf spot, greasy spot or "papaya decline" (spotting of leaves and petioles and defoliation) in St. Croix, Puerto Rico, Florida and Queensland, is caused by Corynespora cassiicola , which is controllable with fungicides.

A new papaya disease, yellow strap leaf, similar to YSL of chrysanthemums, appeared in Florida during the summer in 1978 and 1979.

Black spot, resulting from infection by Cercospora papayae , has plagued Hawaiian growers since the winter of 1952-53. It causes defoliation, reduces yield, blemishes the fruit, and is unaffected by the hot-water dip. It can be prevented by field use of fungicides.

Rhizopus oryzae is most commonly linked with rotting fruits on Pakistan markets. R. nigricans is the usual source of fruit rot in Queensland. Injured fruits are prone to fungal rotting caused by R. stolonifer and Phytophthora palmivora . Stem-end rot occurs when fruits are pulled, not cut, from the plant and the fungus, Ascochyta caricae , is permitted entrance. This fungus attacks very young and older fruits in Queensland and also causes trunk rot. In South Africa, it affects cv 'Honey Gold' which is also subject to spotting by Asperisporium caricae on the fruits and leaves. Both of these diseases are controllable by fungicidal sprays.

Infection at the apex by Cladospoiium sp. is manifested by internal blight. A pre-harvest fruit rot caused by Phomopsis caricae papayae is troublesome in Queensland and was announced from India in 1971. A new disease, papaya apical necrosis, caused by a rhabdovirus, was reported in Florida in 1981.

Papayas are frequently blemished by a condition called "freckles", of unknown origin and mysterious hard lumps of varying size and form may be found in ripe fruits. Star spot (grayish-white, star-shaped superficial markings) appears on immature fruits in Queensland after exposure to cold winter winds. In Uttar Pradesh, an alga, Cephaleuros mycoidea, often disfigures the fruit surface.

In Brazil, Hawaii and other areas, a fungus, Botryodiplodia theobromae, causes severe stem rot and fruit rot. Trichothecium rot ( T. roseum) is evidenced by sunken spots soon covered by pink mold on fruits in India. Charcoal rot, Macrophomina phaseoli, is reported in Pakistan.

Young papaya seedlings are highly susceptible to damping-off, a disease caused by soil-borne fungi– Pythium aphanidermatum, P . ultimum, Phytophthorap palmivora , and Rhizoctonia sp.,–especially in warm, humid weather. Pre-planting treatment of the soil is the only means of prevention.

Papayas generally do poorly on land previously planted with papayas and this is usually the result of soil infestation by Pythium aphanidernwtum and Phytophthora palmivora. Plant refuse from previous plantings should never be incorporated into the soil. Soil fumigation is necessary before replanting papayas in the same field.

Plate XLVII: PAPAYA, Carica papaya
Food Uses

Ripe papayas are most commonly eaten fresh, merely peeled, seeded, cut in wedges and served with a half or quarter of lime or lemon. Sometimes a few seeds are left attached for those who enjoy their peppery flavor but not many should be eaten. The flesh is often cubed or shaped into balls and served in fruit salad or fruit cup. Firm-ripe papaya may be seasoned and baked for consumption as a vegetable. Ripe flesh is commonly made into sauce for shortcake or ice cream sundaes, or is added to ice cream just before freezing or is cooked in pie, pickled, or preserved as marmalade or jam. Papaya and pineapple cubes, covered with sugar sirup, may be quick-frozen for later serving as dessert. Half-ripe fruits are sliced and crystallized as a sweetmeat.

Papaya juice and nectar may be prepared from peeled or unpeeled fruit and are sold fresh in bottles or canned. In Hawaii, papayas are reduced to puree with sucrose added to retard gelling and the puree is frozen for later use locally or in mainland USA in fruit juice blending or for making jam.

Unripe papaya is never eaten raw because of its latex content. [Raw green papaya is frequently used in Thai and Vietnamese cooking.] Even for use in salads, it must first be peeled, seeded, and boiled until tender, then chilled. Green papaya is frequently boiled and served as a vegetable. Cubed green papaya is cooked in mixed vegetable soup. Green papaya is commonly canned in sugar sirup in Puerto Rico for local consumption and for export. Green papayas for canning in Queensland must be checked for nitrate levels. High nitrate content causes detinning of ordinary cans, and all papayas with over 30 ppm nitrate must be packed in cans lacquered on the inside. Australian growers are hopeful that the papaya can be bred for low nitrate uptake.

A lye process for batch peeling of green papayas has proven feasible in Puerto Rico. The fruits may be immersed in boiling 10% lye solution for 6 minutes, in a 15% solution for 4 minutes, or a 20% solution for 3 minutes. They are then rapidly cooled by a cold water bath and then sprayed with water to remove all softened tissue. Best proportions are 1 lb (.45 kg) of fruit for every gallon (3.8 liters) of solution.

Young leaves are cooked and eaten like spinach in the East Indies. Mature leaves are bitter and must be boiled with a change of water to eliminate much of the bitterness. Papaya leaves contain the bitter alkaloids, carpaine and pseudocarpaine, which act on the heart and respiration like digitalis, but are destroyed by heat. In addition, two previously undiscovered major D 1 -piperideine alkaloids, dehydrocarpaine I and II, more potent than carpaine, were reported from the University of Hawaii in 1979. Sprays of male flowers are sold in Asian and Indonesian markets and in New Guinea for boiling with several changes of water to remove bitterness and then eating as a vegetable. In Indonesia, the flowers are sometimes candied. Young stems are cooked and served in Africa. Older stems, after peeling, are grated, the bitter juice squeezed out, and the mash mixed with sugar and salt.

In India, papaya seeds are sometimes found as an adulterant of whole black pepper. Collaborating chemists in Italy and Somalia identified 18 amino acids in papaya seeds, principally, in descending order of abundance, glutamic acid, arginine, proline, and aspartic acid in the endosperm and proline, tyrosine, lysine, aspartic acid, and glutamic acid in the sarcotesta. A yellow to brown, faintly scented oil was extracted from the sundried, powdered seeds of unripe papayas at the Central Food Technological Research Institute, Mysore, India. White seeds yielded 16.1% and black seeds 26.8% and it was suggested that the oil might have edible and industrial uses.

The papaya is regarded as a fair source of iron and calcium a good source of vitamins A, B and G and an excellent source of vitamin C (ascorbic acid). The following figures represent the minimum and maximum levels of constituents as reported from Central America and Cuba.

Food Value Per 100 g of Edible Portion

Fruit Leaves*
Calories 23.1-25.8
Moisture 85.9-92.6 g 83.3%
Protein .081-.34 g 5.6%
Fat .05-.96 g 0.4%
Carbohydrates 6.17-6.75 g 8.3%
Crude Fiber 0.5-1.3 g 1.0%
Ash .31-.66 g 1.4%
Calcium 12.9-40.8 mg 0.406% (CO)
Phosphorus 5.3-22.0 mg
Iron 0.25-0.78 mg 0.00636%
Carotene .0045-.676 mg 28,900 I.U.
Thiamine .021-.036 mg
Riboflavin .024-058 mg
Niacin .227-555 mg
Ascorbic Acid 35.5-71.3 mg 38.6%
Tryptophan 4-5 mg
Methionine 1 mg
Lysine 15-16 mg
Magnesium 0.035%
Phosphoric Acid 0.225%

Carotenoid content of papaya (13.8 mg/100 g dry pulp) is low compared to mango, carrot and tomato. The major carotenoid is cryptoxanthin.

The latex of the papaya plant and its green fruits contains two proteolytic enzymes, papain and chymopapain. The latter is most abundant but papain is twice as potent. In 1933, Ceylon (Sri Lanka) was the leading commercial source of papain but it has been surpassed by East Africa where large-scale production began in 1937.

The latex is obtained by making incisions on the surface of the green fruits early in the morning and repeating every 4 or 5 days until the latex ceases to flow. The tool is of bone, glass, sharp-edged bamboo or stainless steel (knife or raxor blade). Ordinary steel stains the latex. Tappers hold a coconut shell, clay cup, or glass, porcelain or enamel pan beneath the fruit to catch the latex, or a container like an "inverted umbrella" is clamped around the stem. The latex coagulates quickly and, for best results, is spread on fabric and oven-dried at a low temperature, then ground to powder and packed in tins. Sun-drying tends to discolor the product. One must tap 1,500 average-size fruits to gain 1 1/2 lbs (0.68 kg) of papain.

The lanced fruits may be allowed to ripen and can be eaten locally, or they can be employed for making dried papaya "leather" or powdered papaya, or may be utilized as a source of pectin.

Because of its papain content, a piece of green papaya can be rubbed on a portion of tough meat to tenderize it. Sometimes a chunk of green papaya is cooked with meat for the same purpose.

One of the best known uses of papain is in commercial products marketed as meat tenderizers, especially for home use. A modern development is the injection of papain into beef cattle a half-hour before slaughtering to tenderize more of the meat than would normally be tender. Papain-treated meat should never be eaten "rare" but should be cooked sufficiently to inactivate the enzyme. The tongue, liver and kidneys of injected animals must be consumed quickly after cooking or utilized immediately in food or feed products, as they are highly perishable.

Papain has many other practical applications. It is used to clarify beer, also to treat wool and silk before dyeing, to de-hair hides before tanning, and it serves as an adjunct in rubber manufacturing. It is applied on tuna liver before extraction of the oil which is thereby made richer in vitamins A and D, It enters into toothpastes, cosmetics and detergents, as well as pharmaceutical preparations to aid digestion.

Papain has been employed to treat ulcers, dissolve membranes in diphtheria, and reduce swelling, fever and adhesions after surgery. With considerable risk, it has been applied on meat impacted in the gullet. Chemopapain is sometimes injected in cases of slipped spinal discs or pinched nerves. Precautions should be taken because some individuals are allergic to papain in any form and even to meat tenderized with papain.

In tropical folk medicine, the fresh latex is smeared on boils, warts and freckles and given as a vermifuge. In India, it is applied on the uterus as an irritant to cause abortion. The unripe fruit is sometimes hazardously ingested to achieve abortion. Seeds, too, may bring on abortion. They are often taken as an emmenagogue and given as a vermifuge. The root is ground to a paste with salt, diluted with water and given as an enema to induce abortion. A root decoction is claimed to expel roundworms. Roots are also used to make salt.

Crushed leaves wrapped around tough meat will tenderize it overnight. The leaf also functions as a vermifuge and as a primitive soap substitute in laundering. Dried leaves have been smoked to relieve asthma or as a tobacco substitute. Packages of dried, pulverized leaves are sold by "health food" stores for making tea, despite the fact that the leaf decoction is administered as a purgative for horses in Ghana and in the Ivory Coast it is a treatment for genito-urinary ailments. The dried leaf infusion is taken for stomach troubles in Ghana and they say it is purgative and may cause abortion.

Studies at the University of Nigeria have revealed that extracts of ripe and unripe papaya fruits and of the seeds are active against gram-positive bacteria. Strong doses are effective against gram-negative bacteria. The substance has protein-like properties. The fresh crushed seeds yield the aglycone of glucotropaeolin benzyl isothiocyanate (BITC) which is bacteriostatic, bactericidal and fungicidal. A single effective does is 4-5 g seeds (25-30 mg BITC).

In a London hospital in 1977, a post-operative infection in a kidney-transplant patient was cured by strips of papaya which were laid on the wound and left for 48 hours, after all modern medications had failed.

Mention has already been made of skin irritation in papaya harvesters because of the action of fresh papaya latex, and of the possible hazard of consuming undercooked meat tenderized with papain. It must be added that the pollen of papaya flowers has induced severe respiratory reactions in sensitive individuals. Thereafter, such people react to contact with any part of the plant and to eating ripe papaya or any food containing papaya, or meat tenderized with papain.

The mountain papaya ( C. candamarcencis Hook. f.), is native to Andean regions from Venezuela to Chile at altitudes between 6,000 and 10,000 ft (1,800-3,000 m). The plant is stout and tall but bears a small, yellow, conical, 5-angled fruit of sweet flavor. It is cultivated in climates too cold for the papaya, including northern Chile where it thrives mainly in and around the towns of Coquimbo and La Serena at near-sea-level. The fruit (borne all year) is too rich in papain for eating raw but is popular cooked, and is canned for domestic consumption and for export. The plant grows on mountains in Ceylon and South India does well at 1800 ft (549 m) in Puerto Rico. Its high resistance to papaya viruses is of great interest to plant breeders there and elsewhere.

The babaco, or chamburo (C. pentagona Heilborn), is commonly cultivated in mountain valleys of Ecuador. The plant is slender and no more than 10 ft (3 m) high, but the 5-angled fruits reach a foot (30 cm) in length. Usually seedless, or with only a few seeds at most, the fruits are locally eaten only after cooking. The plant is not known in the wild and botanists have suggested that it may be a hybrid. It is propagated by cuttings and is grown on a small scale in Australia and New Zealand primarily for export.


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