the pattern of reproductive development in chenopodium rubrum l

Journal of Experimental Botany, Vol. 27, No. 98, pp. 541-551, June 1976

The Pattern of Reproductive Development inChenopodium rubrum L.

ROBERT EDWARD COOKDepartment of Biology, Yale University New Haven, GT 065201

Received 9 July 1975

ABSTRACT

Individual plants of Chenopodium rubrum were given different numbers of inductive cycles ina 12 h photoperiod and the pattern of reproductive development was analysed after 40 d ofgrowth. At least 2 inductive cycles are required to form any determinate reproductive organsand at least 12 cycles are required for normal reproductive development. Individuals given asingle inductive cycle display a loss of apical dominance at those nodes formed immediatelyafter the treatment without the subsequent formation of any floral structures. Plants givenbetween 2 and 12 inductive cycles display both determinate reproductive organs and indeter-minate vegetative shoots. The pattern of reproductive development on such plants dependsupon the number of cycles relative to the developmental age of newly initiated primordia.It is suggested that the early events of floral induction may involve a radical decrease in theratio of auxin to cytokinin.

INTRODUCTION

As in much of the work with other photoperiodically sensitive species, experimentson floral induction in Chenopodium rubrum have focused on the regulatory processesthat inhibit or promote the creation of a specific floral stimulus in the leaves ratherthan the processes that determine the course of floral evocation at the apex (Evans,1969). Much of this work has utilized seedling populations grown in Petri dishes,with the rate of floral differentiation at the apex of individual seedlings serving as ameasure of the degree of induction (Cumming, 1969). Using this physiologicalcriterion one can determine the minimum number of inductive cycles and theoptimal length of the photoperiod that lead to the most rapid floral differentiationwithin a short time after the start of treatments. One limitation of this procedureis that it obscures the developmental events during organ formation in older plantsand gives little indication of the effects of less than optimal conditions on com-ponents of yield and fitness.

Working with two latitudinal populations of C. rubrum, I have recently examinedthe effects of two different inductive photoperiods on the determination of meanseed weight (Cook, 1975) and on potential seed number (Cook, 1976). The presentpaper examines the effects of different numbers of inductive cycles of standard

1 Present address: Department of Biology, Harvard University, Cambridge, Massachusetts 02138.WITH ONE PLATE IN THE TEXT

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542 Cook—Reproductive Development in Chenopodium

photoperiod on the morphology and reproductive development of individuals froma single population of C. rubrum,. Several interesting developmental events occur inplants given different numbers of inductive cycles and these stress the importanceof meristematic events in regulating the reproductive biology of this species.

METHODSChenopodium rubrum, an annual weed, displays a determinate, ahort-day reproductive responseto inductive photoperiods. Flowers are borne on axillary shoots and are clustered into glomer-ules which become more densely packed (because of lesser axillary intemode elongation) inrelatively shorter inductive photoperiods. A single, relatively larger flower is found at the endof each axillary shoot and at the apex of the main axis.

I obtained seed samples from B. G. Camming of a population located at 50°10' N, 105°35' W(No. 184, Swift Current, Saskatchewan, Canada). The critical photoperiod of this populationis between 16 and 17 h. Seeds were sown in vermiculite-filled Petri dishes and soaked withdistilled water. The dishes were placed in a constant temperature (23 °C) growth chamber with18 h photoperiods (13 000 lx of 9 :1 fluorescent: incandescent illumination) and 6 h dark periodsfor five 24 h cycles. During the first and second dark period seeds were chilled at 5 °C, a treat-ment which increased germination By the fifth day seedlings had open cotyledons and wereready to be transplanted to vermiculite-filled 3 inch plastic pots. Two experiment growthchambers were used to give the plants inductive treatments: an 18 h light :6 h dark photo-period chamber in which the plants remained vegetative and a 12 h light: 12 h dark photo-period chamber in which floral induction rapidly occurs. It was not possible to give vegetativeand reproductive plants the same total amount of light and possible photosynthetic effectswere judged to be minor relative to the total pattern of reproductive development. All potswere irrigated with nutrient solution (Hyponex 7-6-19, Hydroponics Chemicals Co., Copley,Ohio; 1-2 g 1-1) four times daily and the solution was renewed once each week.

A total of 156 seedlings were planted in plastic pots and placed in the 18 h chamber. After15 d of growth in this non-inductive photoperiod, 6 plants were dissected and the number ofleaves and leaf primordia above the cotyledons were counted. All dissections were carried outunder a binocular dissecting microscope. Plants averaged 10 ± 1 leaves or leaf primordia at thestart of the experiment. To begin treatments groups of 6 plants were given different numbers ofinductive cycles in the 12 h photoperiod. Thus 144 plants were placed in the 12 h photoperiodand 6 control plants remained in the 18 h chamber. On successive days groups of 6 randomlyselected plants were returned to the 18 h chamber to continue growth or complete reproductivedevelopment. At the end of 40 d of growth (after the start of treatments) all plants were ex-amined to assess the pattern of reproductive development. Thus, at least 6 plants had receivedeither 0, 1, 2, 3, 4, . . . 19, 20, 24, 28, 32, or continuous inductive cycles.

RESULTS

Individuals of Chenopodium rubrum given continuous induction differentiateflowers at all axillary and apical meristems and no additional vegetative leaves orshoots are formed. After 40 d of growth seeds have matured and most leaves aresenescing or dead. All plants given 13 or more inductive 12 h cycles were indis-tinguishable from plants given continuous induction (Fig. 1). A total of 25 nodeson the main axis were fully reproductive with mature seeds and senescent leaves.Because the seeds originally came from a wild population, there is some variationbetween individuals in the exact duration of reproductive development. This alsoapplies to the pattern of reproductive development among individuals givendifferent numbers of inductive cycles. The variation is small, however, relative tothe larger pattern and usually involves the relative timing of developmentalevents. The description given below should be considered typical rather thandefinitive.

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Cook—Reproductive Development in Chenopodium 543

An understanding of the effects of different numbers of inductive cycles on thedevelopmental pattern of C. rubrwm conies most easily when the normal course ofdevelopment is reviewed (Cook, 1975). The initial effects of induction observed onall apical and axillary meristems are an increase in the rate of growth, a decreasein the plastochron and the simultaneous growth, and precocious development ofaxillary buds leading to the well known double-ridge appearance. On the main axisthis enhanced rate of organ initiation begins 2 d after the start of induction in a12 h photoperiod and continues for 4 d. On the 6th day the terminal meristemdifferentiates a floral ridge and no new primordia can be initiated on the main axis;the number of nodes becomes fixed. At this time close inspection of the apex revealsthat precociously developing axillary buds have not yet differentiated a floral ridge.Thus the sequence of floral differentiation is basipetal. This general pattern ofreproductive events at the apex of C. rubrum is essentially similar, except forimportant differences in timing, to that seen in C. amaranticolor (Thomas, 1961),C. album (Gifford and Tepper, 1961), and in many other species (Langer and Bussell,1964). The important points are a promotion of the rate of organ initiation followedby an inhibition coincident with the differentiation of a terminal flower.

Single cycle inductionAll individuals given a single inductive cycle remained completely vegetative

and close examination revealed no organs that could be interpreted as reproductivestructures; all newly initiated primordia developed as leaves and the plants con-tinued to grow vegetatively. However, the single inductive cycle was not withouteffect upon subsequent growth and form. When examined 40 d after treatment, theaxillary buds at those nodes formed during and after the single inductive cycle(nodes 11-20) had initiated growth and developed into vigorous vegetative shoots

T A B L E 1. The initiation and growth of axillary shootsPlants of C. rubrum were grown for 15 d in LD (18 h photoperiod) before being given a singlecycle in a 12 h photoperiod and returned to the non-inductive photoperiod for 40 d. The controlplants remained in the non-inductive photoperiod for 56 d. Each node was dissected and thenumber of primordia on the axillary shoot and its length were measured. Each measurementrepresents the mean of 6 individuals.

Node abovecotyledons

101214161820222426283032

Length of shoot

1 cycle

—

14-17 (± 2-71)63-00 <± 7-85)79-50 (± 9-18)73-00 (±14-00)55-67 (±11-48)42-67 (± 916)33-33 (± 8-66)22-50 (±10-60)10-17 (± 6-15)3-50 (± 2-07)0-33 (± 0-52)

(mm ±s.d.]1

LD control

—

7-83 (±22-33 (±

7-52)9-35)

20-50 (±10-95)13-50 (±24-83 (±10-83 (±6-67 (±4-00 (±2-83 (±1-50 (±

—

6-57)4-26)5-12)3-50)2-97)2-99)1-76)

Number of primordia (±s.d.)

1 cycle

17-33 (±1-21)24-50 (±2-88)36-33 (±2-07)40-50 (±1-52)3917 (±1-72)36-83 (±1-72)35-33 (±1-21)33-83 (±1-33)32-00 (±1-55)29-17 (±1-17)26-33 (±1-86)24-17 (±3-31)

LD control

15-67 (±1-51)21-00 (±2-61)27-00 (±3-16)29-33 (±2-73)29-50 (±3-15)29-50 (±3-15)29-00 (±2-48)27-67 (±2-88)27-67 (±3-98)26-83 (±4-58)26-00 (±4-10)25-17 (±5-64)

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clearly larger than those at the same nodes on plants kept continuously in 18 hnon-inductive conditions. (Table 1; Plate 1H, J ) .

The axillary shoots at every other node on the 6 plants which received thistreatment were dissected, as were the 18 h non-induced controls, and the number ofleaves or leaf primordia and the length of the shoot were quantified. These resultsare shown in Table 1. Clearly the axillary shoots at these nodes have initiatedvigorous growth. It should also be noted in Plate 1 J that the growth of the axillaryshoots has carried the subtending leaf at this node away from the main axis of theplant. Recent work by Sachs (1970) has shown that the lateral buds released fromapical dominance by the excision of the shoot induce the differentiation of directvascular contacts to the strands of the cut stem. Such connections are normallyprevented by the flow of auxin in intact plants and growing lateral buds are con-nected to the roots by vascular strands which run parallel to the vascular system ofthe stem. Plate 1J suggests that the primary vascular connections of the growinglateral shoot are to the vascular strands of the leaf. Thus a single cycle may involvechanges in the production of auxin by newly initiated leaf primordia. This inter-pretation is supported by the general observation that induction is correlated witha decrease in the auxin status of the shoot (Beever and Woolhouse, 1975). Furtheranatomical studies are presently being conducted.

Multiple cycle inductionAH plants given 2 or more inductive cycles differentiated some reproductive

structures. The form of these structures and the pattern of their development onthe plant depended upon the number of inductive cycles given (See Plate 1 and itsdescription). Most individuals given between 2 and 12 inductive cycles formed bothfloral, determinate inflorescences and vegetative, indeterminate shoots thatcontinued to initiate new leaf primordia. The location of these vegetative shoots ormeristems depended upon the number of cycles given.

In addition these individuals displayed numerous growth anomalies which rangedfrom foliar structures with curled lamina and twisted petioles to undeveloped floralstructures with enlarged and distorted bracts, rudimentary stamens, and elongatedpeduncles (Plate ID—G). Most of these reproductive anomalies appeared to haveceased growth and differentiation at an early stage of development. Those plantsgiven a greater number of inductive cycles had fewer such organ anomalies andthose that were present had achieved a greater degree of development toward eitherreproductive or vegetative structures before the normal course of differentiation wasdisturbed. Both determinate reproductive organs and vegetatively growing meri-stem s could be found in close proximity on the same shoot. Dissection revealed that onall such plants there were some meristems that had escaped evocation and whetherthey were growing or not depended upon the number of cycles given. Plants given10, 11, or 12 cycles displayed fasciations of bracts, undeveloped leaves, floral struc-tures, and dormant vegetative buds (Plate 1G, K) in a mass of uncoordinated growth.

It should be noted that the fasciation of undeveloped buds seen in Plate lGresembles the generation of numerous buds caused by pathogens such as thebacterium Corynebacterium fascians in a variety of dicotyledonous plants (Thimann

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and Sachs, 1966). These authors were able to imitate the pathology through theapplication of kinetin and they were able to isolate an active cytokinin from infectedtissue, suggesting that the fasciation was caused by the bacterial synthesis of cyto-kinin. The appearance of similar symptoms on plants of C. rvbrwm given differentnumbers of inductive cycles likewise suggests that the relative concentrationof cytokinin may be greatly increased by induction.

The pattern of whole plant developmentThe pattern of partial reproductive development of the whole plant displayed in

Fig. 1 can be most meaningfully understood in terms of the age of newly initiatedprimordia. The first important fact is that individual primordia and axillary buds

25

20

15

|

10

LD 1 cycle 2 cycles

o1

"I

3 cycles

25

20

15

|

10

4,5, or 6cycles

7,8, or 9cycles

10,11 or 12cycles

13 or morecycles

FIG. 1. A diagrammatic representation of the pattern of reproductive development inplants of O. rubrum given different numbers of inductive cycles and examined 40 d after thestart of treatments. Plants were grown for 15 d in a non-inductive (18 h) photoperiod andgiven the different treatments in an inductive (12 h) photoperiod after which they werereturned to non-inductive conditions. At the start of treatments each individual averaged10 leaves or leaf primordia on the main axis. Symbols: A, vegetative indeterminate shoot;O, reproductive determinate shoot; Q, undeveloped floral primordia and fruits; • , mature

seeds characteristic or normal reproductive development. LD = 18 h photoperiod.

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respond differently to the same number of inductive cycles, and the developmentalresponse depends upon the position or age of the bud relative to the start and dura-tion of inductive treatments. No vegetative axillary shoots were seen to be growingat those nodes (1-10) formed before the start of induction; all meristems on theseaxillary shoots were determinate and reproductive. Generally, for any given numberof inductive cycles, the degree of reproductive development was greater at theseolder nodes relative to higher (younger) nodes on the plant.

This age-dependent interpretation of meristem determination is supported bythree observations. First, individuals given only two inductive cycles differentiatedreproductive structures on those axillary shoots at nodes that were already formedat the start of induction (i.e. nodes 1-10); the terminal meristems of these shootsdisplayed floral organs in various stages of development. The apical meristem andall axillary shoots that began growth after the start of treatments were completelyvegetative and were vigorously growing, much like those of plants given a singleinductive cycle.

Second, all individuals given 3, 4, 5, or 6 inductive cycles differentiated bothvegetative and reproductive structures at every node. (Plate 1A-F). The floralstructures found at each node were usually structural mixtures, showing unde-veloped characteristics of flowers as well as distorted vegetative characteristics.In addition to undeveloped floral structures, those nodes formed immediately afterthe start of induction (11-20) usually had two such shoots separated by a floralstructure (Plate 1 A , B). These vigorously growing axillary shoots escaped both apicaldominance and reproductive determination despite the clear differentiation of otherreproductive organs beside them. I interpret the pattern of growth at such nodesas representing an axillary shoot whose terminal meristem has differentiated intoa determinate floral organ while two younger second-order axillary buds have beenreleased from apical control but have also simultaneously escaped floral evocation.

Third, those nodes formed immediately after the start of induction (11-19)displayed the most vigorously growing escaped axillary shoots and, for any giventreatment, the degree of normal reproductive development increased at higher(younger) nodes. For example a plant given 9 or 10 inductive cycles might displaynormal reproductive development on axillary shoots at nodes 1-10 and at the apexof the plant (nodes 22—25) while various forms of disturbed growth appeared onaxillary shoots at nodes 13—17. Plate IK represents a plant whose reproductivedevelopment was apparently normal at every node except node 20 (out of 27 on theplant) where a massive fasciation of undeveloped buds was growing. It is those nodesinitiated immediately after the start of induction that are also the most likely nodesto have initiated the second- and higher-order (i.e. younger) axillary buds which canescape evocation and continue vegetative growth.

This suggests that the developmental state of newly initiated meristems maydetermine whether evocation takes place or not. Clearly the leaf-bound processes ofinduction have been sufficiently initiated by 2 inductive cycles to lead to thedifferentiation of floral structures on lower axillary shoots. An additional 10 cyclesare required to determine all axillary meristems, particularly those that begingrowth after the start of inductive treatments. The growth and permanent escape

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from floral induction of certain axillary meristems in close proximity to othersthat have undergone floral differentiation argues that age and not position deter-mines whether the floral stimulus will be effective. If the process of evocation isinterrupted by a return to non-inductive conditions, these younger, growingmeristems escape evocation and continue vegetative growth. This leads to thepattern of reproductive development seen in Fig. 1.

The differential response of meristems depending upon their age may involve thetiming of the division cycle in the cells of the meristematic tissue relative to thepresence of the inductive stimulus (I. Sussex, personal communication). It appearsthat in some species the cells of inhibited axillary buds are all arrested in Gl (Yunand Naylor, 1973) and further DNA synthesis and cell division begins when apicaldominance is removed. Such older axillary buds may require fewer inductivecycles because all of their cells are synchronized and ready for DNA synthesis anddifferentiation. The newly initiated meristem would appear to pass through a periodof early growth when it cannot be determined as a reproductive shoot despite thepresence of sufficient floral stimulus to differentiate nearby but older meristems.If non-inductive conditions return before this period is complete, evocation will notoccur and these meristems will continue growth and leaf initiation. Such growthprobably inhibits the further reproductive development of already determinedfloral meristems in close proximity. The very youngest meristems present after thestart of induction will be the second- and higher-order primordia, and I believe theseare the vigorous shoots seen in Fig. 2. A tentative hypothesis is that this period ofinsensitivity is related to the duration of the cell cycle in the cells of newly initiatedmeristems.

In addition this period of insensitivity may determine the minimum number ofinductive cycles required for floral evocation. In a recent paper examining therequirement for a minimum number of inductive cycles in Perilla and Xanthium,Jacobs (1972) has hypothesized that full floral induction required as many 24 hinductive cycles as there are hours in the vegetative plastochron of the speciesinvolved. In this population of G. rubrum grown under the present conditions, thevegetative plastochron is 0-44 d (Cook, 1976). In the experiment reported here atleast 2 inductive 12 h cycles are required to differentiate any floral structures onlower axillary shoots, and 3 cycles were required to differentiate a floral ridge at theapex. It appears that the minimum number of cycles required may depend more onthe rate at which newly initiated primordia differentiate as determinate organs andassociated meristems become organized into growing structures. Because of thenecessarily correlated control of growth within the plant, this rate is generallyrelated to the plastochron.

CONCLUSIONSThe essential observations are these.

(1) A single inductive cycle leads to developmental changes at the apex withoutthe determination of any reproductive structures.

(2) A six-fold greater number of inductive cycles are required to determine all

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meristems as reproductive than are required to determine a single meristemas reproductive.

(3) Second-order axillary meristems are able to escape evocation and remainvigorously vegetative despite the reproductive determination of othermeristems on the same axillary shoot.

(4) Meristems present before induction require fewer inductive cycles to achievea given degree of reproductive development than those meristems initiatedafter induction.

(5) The inhibitory effect of non-inductive cycles on the development of repro-ductively determined meristems appears correlated with the presence ofundetermined vegetative meristems growing on the same individual.

(6) The developmental fate of a meristem depends upon its age relative to thestart and duration of induction.

The loss of epical dominance in newly initiated axillary buds of plants given asingle inductive cycle indicates that some stimulus produced in the leaves hascaused a developmental response in the apical meristem of the plant without sub-sequent floral differentiation. This suggests that the control of floral evocation lies,at least in part, with the developmental and metabolic state of the axillary andapical meristems. It also cautions against interpreting the failure of plants todifferentiate floral structure when given less than the minimum number of inductivecycles solely in terms of a failure to synthesize the floral stimulus in the leaves. Inone cycle with plants of C. rubrun induction has occurred in the leaf but floralevocation has not. The evidence also suggests that the very earliest events of floralinduction involve relative changes in auxin and cytokinin, and the floral evocationof individual primordia may depend upon the internal timing of determinationrelative to these changes.

ACKNOWLEDGEMENTSI would like to thank Drs. Ian Sussex, Tsvi Sachs, and my thesis advisor, ArthurGalston, for help throughout this work and for critically reading the manuscript.This work was done in partial fulfillment, of the requirements for the Ph.D. in theDepartment of Biology at Yale University. During this time I was supported by anN.S.F. Traineeship.

LITERATURE CITEDBEEVEB, J. E., and WOOLHOUSE, H. W., 1973. Nature (New Biol.), 246, 31-2.

1975. J. exp. Bot. 26, 451-63.COOK, R. E., 1975. Am. J. Bot. 62: 427-31.

1976. Photoperiod and the determination of potential seed number in Chenopodiumrvbrum L. Ann. Bot. 40.

CUMMTNO, B. G. 1969. In The induction of flowering. Ed. L. T. Evans. Cornell University Press,Ithaca, New York.

EVANS, L. T., 1969. In The induction of flowering. Ed. L. T. Evans. Cornell University Press.Ithaca, New York.

GnTOBD, E. M., and TEPPEB, H. B., 1961. Am. J. Bot. 48, 657-67.JACOBS, W. P., 1972. Ibid. 59, 437-41.LANGEB, R. H. M., and BussEii, W. T., 1964. Ann. Bot. 28, 163-7.

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SAOHS, T. 1970. Israel J. Bot. 19, 484-98.THTAIANN, K. V., and SACHS, T., 1966. Am. J. Bot. 53, 731-9.THOMAS, R. G., 1961. Ann. Bot. 25, 138-51.YTOT, K-B., and NAYLOB, J. M., 1973. Can. J. Bot. 51, 1137-45.

EXPLANATION OF PLATEPLATE 1

Developmental anomalies at selected nodes on plants given between 2 and 12 inductive cyclesin a 12 h photoperiod. Symbols: s = stem or main axis; p = petiole of the leaf at that node;v = vegetative meristem; f = determinate floral organs; a = axillary shoot; tf = terminalflower on the main axis; mf = mature fruits with seeds.A. Node 19 of a plant given 3 inductive cycles. Note the two escaped axillary shoots (v) which

are interpreted as second order axillary buds. The determinate floral structure (f) representsthe terminal meristem of the primary axillary bud.

B. Node 24 of a plant given 3 inductive cycles. Again note the second-order axillary shoots thatremain vegetative despite the differentiation of the primary axillary meristem as a floralorgan (f).

c. Tip of an axillary shoot at node 23 of a plant given 3 inductive cycles. Note the differentiatedfloral structure, one bract of which has differentiated as a leaf.

D. Node 18 of a plant given 7 inductive cycles. Note the degree of vegetative growth on the twoaxillary shoots and compare with F, the same node on a plant given one less inductive cycle.Alan note how the axillary shoots have differentiated a shoot-like internode connecting theleaf petiole (p) with the main stem (s).

E. Node 9 of a plant given 6 inductive cycles. Note the degree of differentiation of the floralorgans (f) and compare with F, a younger (higher) node on a plant given a similar number ofcycles.

F. Node 18 on a plant given 6 inductive cycles. Compare with D, the same node on a 7-cycleplant, and E, a younger node on a 6-cycle plant.

G. Node 16 on a plant given 12 inductive cycles. Some of the fruits in this picture are matureand others remain undeveloped. All meristems appear to have differentiated as floralorgans.

H. Node 12 of a plant grown for 56 d in a non-inductive 18 h photopenod. Note the degree ofgrowth of the axillary shoot and compare with j .

j . Node 12 of a plant grown for 15 d in a non-inductive photoperiod, given a single inductivecycle, and returned to the non-inductive (18 h) photoperiod for an additional 40 d. Com-pare with H. Note how the released axillary shoot has grown out from the main axis (s) andcarried the leaf petiole with it, suggesting vascular connections to the petiole vascularstrands. A new axillary bud can be seen in the axil between the shoot and the petiole.Compare with D where two axillary shoots have connected to the petiole vascular strands.

K. Node 21 of a 10-cycle plant. This individual exhibited completely normal developmentexcept for the presence of the fasciation of primordia at node 21. Note the character of themature fruits and seeds at higher nodes and compare with the undeveloped floral organs (f)characteristics of the fasciation.

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coo K—Reproductive Development in Chenopodium

PLATE 1

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the pattern of reproductive development in chenopodium rubrum l

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