The Biology of Poor Seed Production in Tephrosia Vogelii

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NATlON~l BUREAU 0; STANDARDS·1963.A NATIONAL BUREAU OF STANDARDS-1963-A US3 -/ :it 141 Of TIlE BIOLOGY 'OF ;t POOR SEED PRODUCTION IN TEPHROSIA VOGELII

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~ . NOT LOAN >. ,Y ... ~ Teclmical Bulletin No. 1419 >t .....D d a ~ r- c.;I 0 en :.= E-4 .D '. .... co .. rn ~= 0 ~ ~ ~

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Agricultural Research Service UNITED STATES DEPARTMENT OF AGRICULTURE

,, " Contents

Pap .• Summary . .,.- ",. - ... _-- .. - ~------1 Introduction _._------1 Floral biology . ~---~------~------2 Morphology and life cycle of the flower ______2 Pollen development, abo.rtion, and germination .. ______4 Self-pollination 7 Role of insects in pollination ... _ .. ______. 10 Abscission, pod drop, and pod shape 13 Effects of hormone treatments 15 Pod and seed yields 17 Factors affecting seed yield ... -- ... ___ r_ ...... _,. ___ _ _ 17 Effects of weather changes 18 Correlation of climatic variables with pollen production and self-pollination . ______. 24

Effects of modified environments 25 Varietal and species comparisons 29 ~ j 1 Discussion ! - ~-.- ... ~, ... _"'------.. 31 ~. Evolutionary status of Teph1'osiu vogelii 31 Causes of poor seed production 32 Recommendations for seed production and further study.. 33 Literature cited 34

Washington, D.C. Issued April 1970 For sale by thc Supct"intendcnt of Documents, U.S. Government Printing Office Washington, D.C. 20402 - Price 20 cents

".. .. 1 THE BIOLOGY OF POOR SEED PRODUCTION IN TEPHROSIA VOGELII

By FRANKLIN W. MARTIN, plant geneticist, and EUGENIO CABANILLAS, agri~ultural 1'esearch technician, Crops Reseu1'ch Division, Agriculturc:~ Resea1'ch 3e1'vice SUMMARY Tephrosia vogelt:'i Haak. f. is a patential saurce af the insecti cide ratenone. This plant flawers well, but sets pods and seeds paarly in Puerto. Rica. Variaus factars cause paar fert!Jity, princi pally pallen abartian and failure of anthers to dehisce. Abartion is assaciated with weather canditions and may result if moderate temperatures and high humidities do nat accur during the week . preceding anthesis. Althaugh self-pallinatian is aften accomplished without exter nal agents, large bees increase the amaunt af self-pollination by changing the relatianship af pallen and stigma. In additian, bees may passibly damage the stigmatic surface and thus make it mare receptive to pallen germinatian. The stigma is seldom expased and rarely crass-pallinated. Insect activity may limit seed praduc • tian. The fertilized avary may abscise before maturity, princi pally during the early part af the flawering season, but also dur ing hat, very dry weather later in the season. Harmone treat ments benefit pad and seed set when abscissian is a prablem. Minar causes af paar seed set include floral abnarmalities, in sect damage, and paar flawer develapment near the <,md of the f1awering seasan. A caal, maist enviranment, high nutritional levels, and insect cO"ltrol measures may maximize seed productian. Varietal differ ences in fertility suggest that seed praductian can also be in creased thraugh breeding.

INTRODUCTION Plants af the genus Tephl'osicL frequently cantain ratenaid com paunds useful far killing insects and undesirable fish (9).:1 Hawever, few species contain enough rotenone to justify their use as insecticide crop plants (5). Because of its high rotenone content, large size, rapid growth, and adaptation to some re gions of the United States, Teph?'osia vogelii Hook. f. has been se lected for study and for development as a new crop species. Stud ies of this species in Puerto Rico showed that mature plants flow ered as day lengths decreased during the fall (6). However, in most parts of the United States plants do not mature sufficiently or are killed by frost before flowering. Therefore, although T. vo

l Italic numbers in parentheses refer to Literature Cited, p. 34. 1

It 2 TECHNICAL BULLETIN 1419, U.S. DEPT. OF AGRICULTURE gei:i;i can be grown as an insecticide pla."!lt in tho continental United states, it requires a tropical location for sefid production, as well as for breeding and improvement (1). ~., During the Tephrosw, breeding program at the Federal Experi ment Station in Puerto Rico, investigators observed that plants bloomed freely but set seed poorly. Various aspects of this seed setting problem had been investigated, but the results were incon , clusive and were not published. In 1965 .more extensive studies on the reproductive processes of T. vogeli-i were undertaken to deter I ~ mine the principal causes of poor seed set. The results of these ! investigations are summarized here. FLORAL BIOLOGY Morphology and Life Cycle of the :Flower The typical flower of the family Papilionaceae (Fabaceae) is hermaphroditic and strongly zygomol'phIc. One petal, the stand ard, is often enlarged and vertical. The two lateral petals are usually extended as wing3. The two lower petals are united into a structure called the keel, which envelops the stamens and the pis til. The stamens are partially or entirely united in a tube sur rounding the ovary (4). Flowers are adapted to either of two kinds of breeding systems. In self-pollinated species (soybeans, lespedeza, some field beans) the pollen is usually shed directly onto the stigma before or as soon as the flower opens. In cross-pollinated species (clover, al falfa) various agents, chiefly insects, promote exchange of pollen. Self-fertilization of such species is often impeded by full or par tial seif-incompatibility. Three typical means of pollen dispersal include exposure of anthers and stIgma, pumping- of pollen from a cavity in the keel to the outside with the pistonlike action of the stamens, and explosive release of pollen coupled with exsertion of the staminal column. All these mechanisms depend on the depres sion of the keel by visiting insects (8). The flower of Teph7'osia vogel'ii is typical of the Papilionaceae (fig. 1). It is large, with a standard 2.5 cm. high and 3 cm. wide, keel and 'wings ~ cm. long, and wings extending 1.8 to 2.4 cm. to the sides. The flowers are borne in compact racemes of 50 to 200 flowers, which bloom over a 3- to 6-week period. Each flowering .L node bearing two flowers is subtended by a fugacious bract that leaves a scar. The flowers are either purple with white markings or entirely white. . Anthesis of the normal flower may begin at any hour but is more frequent during the mornhg. It begins as a splitting of the suture of the standard below the keel. About the time that the standard unfolds, the anthers dehisce. Usually dehiscence is com plete by the time the standard has fully expanded. However, one or more anthers may fail to dehisce. They do not dehisce later. The pollen grains are somewhat glutinous and are aggregated to gether. After a short time these aggregations break up and indi vidual grains fall. ~ " •.Jr...... :" .," ",.,..... ,...... '. ;(-.: -."...."', .. . "..,..' .~ ... '" '" .'

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B:-:-35490, BN-35493, BN-45491, BN-35492 FIGURE I.-Flower of Tephrosia vogelii: A, Side view of newly opened flower; B, variations in keel structure' (anthers caught in upper suture of one flower); C, anther position and stamen length in old, intermediate, and young flowers; V, normal style (left) and three abnormal styles. x 1.25. <:0 4 TECHNICAL BULLETIN 1419, U.S. DEPT. OF AGRICULTURE

Certain abnormalities of the flower occur at low frequency (1-5 percent). These include failure of the banner to open, inclu sion of a wing in the keel, and adhesion of the upper edges of the ~.' wings. Other more s17btle, more frequent, and probably more im portant abnormalities also occur. The stigma is sometimes so tightly enfolded by the :.:eel that self-pollination is impossible. The style is sometimes bent at an angle of 45° to 90° so that the stigma is not among the anthers. The .:lffect of these abnormali ties is to reduce the chances of self-pollination and in some in stances to make cross-pollination virtually impossible. During 1966 these abnormalities tended to vary in intensity as the flower ing season progressed (table 1). However, the percentage of ab nOlmal flowers was never high enough to account for all the poor pod set. As the flower ages, the upper suture of the keel may split. Less frequently the short leg of the L-shaped keel ruptures. The floYver begins to fade. The two wings become appressed to the keel. As wilting occurs, abscission also takes place. The life of an unpolli natedflowel' is 48 to 56 hours during the rainy season. However, in dry weather flowers wilt and abscise in 30 hours (table 1). If the pod is set, the flower usually does not abscise. The elongating ovary ruptures the wilted keel the third or fourth day after polli nation. One week after anthesis the pod is 2.5 to 5 cm. long, and in 3 weeks it is mature and 7 to 13 cm. long. Fertilized and unfer tilized ovules can be distinguished by size at about 7 days after anthfJsis. The pods mature, dry, and eventually dehisce.

Pollen Development, Abortion, and Germination The chromosome number of T. vogelii is 22, the most common number in its genus (8). The genus is characterized by regular chromosomal behavior. In five typical, partially sterile lines stud ied, the chromosome number was 22. At meiosis of the pollen mother cells, most of the chromosomes paired as bivalents. Occa " sionally, however, multivalents of three and four were found. In about 5 to 10 percent of the cells there were one or two unpaired chromosomes at the metaphase stage. Occasionally one or h.;'o chromosomal pairs, or multivalents, would separate tardily at au aphase, but no signs were found of bridge-and-fragment behavior. No abnormal behavior was observed that could have led to pollen abortion. Pollen abortion,. as measured by small pollen grains and poor stainability, was common in each of 10 Teplw·osia. lines studied. It varied from 5 to 100 percent according to flowers, days, and years. If; was high in 1966 and 1967 but particularly low in 1968. Whell abortion was high, anthers frequently failed to de hisce. The percentage of flowers in which dehiscence occurred varied from day to day (table 2). The abortion was not associated with any particular anther or position of anther in the flower. Extreme pollen abortion led to complete break up of the cells

" ~ •••w.'w... _ .•• •• w .. w.. .'~ .,. . .. "Ji'W~ ..,'W''. · ."" '''. ,s' ." 8 TABLE l.-Seasonal observations of some 'rephrosia floral ~ chamcte1-istics,1966 ~ I:'J 0 Anthers Suture Ends of keel Stigtrtas Abnormal ii.ife of '1:l Date Flowers outside of keel ~ 1 damaged: flowers examined flower 1 open; open styles • 0 0 Number Number Number Number Number Number Hours c: 0 October 2 48-56 4 52 14 40 0 2 ~ 6 0 1 48-56 w 32 0 11 - .. ~ ~ . 50 18 7 38 0 0 0 48-56 ~ 18 - ~ - ~ - ....- 50 37 1 0 0 48-56 ..... 25 - •." ...... ~ ~ 50 6 W November Z 50 6 28 1 0 0 48-56 ...,..... 1 48-56 "'3 8 50 11 18 0 1 2 I:'J I 16 1 0 0 30-36 '1:l 15 ., "" ... 50 3 -' - - """ 2 0 1 30-36 p:: 22 - .. * ...... 50 7 13 0-... ~ 1 'Result of insect activity. g : Impede self- or cross-pollination. >..... 8 ~ .....

C11 6 TECHNICAL .BULLETIN 1419, U.S. DEPT. OF AGRICULTURE

TABLE 2.-Propodion of matu,re T. vogelii /lowers with so.?ne pollen on stigma, as affected by sta'men length, and proport':on 4 of /lowe1's 'with unopened anthers, 1966 .. Long stamens with- Short stamens with- Flowers with Date Pollen 011 No pollen Pollen on No pollen unopened stigma on stigma stigma on stigma anthers Percent Percent Percent Percent Percent October i~ 4 -00\_ ... - __ " 60 8 10 22 18 , 11 -_ .. -...... 64 E 12 16 38 i"., 18 ..... -.. _- ... - 61 12 12 26 14 25 ...... 76 4 6 14 8 Novemb(!r 1 -- .. _.... ,.. .. 82 4 4 10 6 8 .. ,...... "" ... 80 8 6 8 0

16 ... ", .. -.. " -'" 84 4 4 8 4 22 ." ... ____ w_ 78 8 10 4 0

1 and l~elease of large quantities of particles. These particles could i I not be cultured in media suitable for many micro-organisms and .4 were probably plastids or mitochondria. ~ Pollen abortion evidently began soon after meiosis of the pollen .. i mother cell, based on observations of bud development. Mature and maturing buds due to open in 2 to 3 days hung downward (fig. 2). Transitional or intermediate buds 4 days before anthesis were perpendicular to the stem. Younger buds remained upright in a tight duster. Aborted pollen could be detected clearly in up right buds 5 to 8 days before anthesis. Abortion of the developing pollen of such young buds ranged from 5 to almost 100 percent. A study of buds of various ages suggested that no new abortion be gins after about the fourth day before anthesis. Percent pollen germination in a sucrose and mineral solution recommended by Brewbaker and Kwack (2) varied widely. Pollen germinated best when the sucrose solution varied from 10 to 30 percent. Normal appearing pollen from anthers showing high abortion sometimes did not germinate in vitro. In vivo pollen germination was measured with and without hand pollination by means of an ultraviolet: technique (7). Al though pollen grains on the stigma and pollen tubes within the stigma usually could be detected, pubescence of the style and ovary impeded observations in those organs. This difficulty was partially oven'ome by dissecting the style. Stigmas self- and cross-pollinated by hand in the field invariably contained many ponen tubes, and these could be traced through most of the style. Thus, pollen abortion is highly variable. When adequate pollen is available, germination in vitro and in vivo is excellent. How ever, when pollen abortion is high, lack of viable pollen limits seed production. POOR SEED PRODUCTION IN TEPHROSIA VOGELU 7

• nN-36494 ~ FIGURE 2.-Inflorescence of Tephrosia, showing young and intermediate buds and flowers.

Self.Pollination The flower of T. vogelii appears to be ideally suited for self pollination. In most flowers the stamens extend beyond the stigma and pollination occurs on dehiscence of the anther. In other flow ers, however, the anthers are positioned at or just below the stigma, and self-pollination is impeded. This variation in relation of anther level to stigma level is due almost entirely to differences ,. in length of the staminal tube. Styles in newly opened flowers are uniformly 1 cm. in length, whereas those of mature flowers are only slightly shorter (when measured from the point of curva ture of the style). Stamens that are very short usually bear an thers that do n(;.t l1ehisce. After dehiscence of the anthers, the sta mens shrivel -and thus withdraw the anthers from the area of the stigma. The bulk of the pollen remains in the cavity of the keel around the style. Data from several sources suggest that self- l'ather than cross pollination generally occurs in T. vogeZii. Varieties are uniform and retain their distinguishing characteristics from season to sea son. White-flowered varieties grown next to purple-flowered ones (purple flower is genetically dominant to white flower) have never been known to produce purple-flowered offspring. Pollen germi nates readily on the stigma of its own flower. Flowers bagged be fore anthesis frequently produce pods and seeds. The flowers of 8 TECHNICAL BULLETIN 1419, U.S. DEPT. OF AGRICULTURE plants isolated in cages may produce normal seed (table 6) . Sin gle varieties in isolated plots set seed as well as do several vari eties in mixed plots. In a study of pod set in variety P.I. 257533 under carefully con trolled conditions, 25 pollinations were made daily during most of the flowering season. The pod and seed production of this variety and of open-pollinated controls were compared. Only buds begin ning to open were used for pollination. Buds were opened and the stigmas were pricked with a sharp needle and pollinated with pollen from selected mature flowers, apparently containing large quantities of viable pollen. Afterward the abscission joint of the pedicel and the receptacle of the flower were treated with a mix tlU'e of 0.8 percent indole-3-butyric acid and lanolin. The result ing pods and seeds were counted. Fair but variable pod and seed set were obtained by hand polli nation (table 3). During the early part of the season when natu Tal pod set was low, hand pollination and hormone ti.'eatment in creased pod and seed set. Later, when natural seed set in(!reased, hand pollination and hormone treatment appeared to have little effect. These results suggest that during the early part of the sea-

TABLE J.-Amowzts of pods and seeds p'l'od~wed from 25 hand-poz,. 41I linated, IWl'mone-b'eated Tephrosia flower's, as compared 'with i those fl'o'n~ open-pollinated flowe/'s, and p'roport1;on of flowers with 'unopened anthel's, 1967 .'

Pods Seeds Flowers with Date Hand Open Hand Open unopened pollinated pollinated pollinated pollinated anthers Number Number Nmnbe1' Nlt1nbe1' Pe1'cent October 9 14 1 87 16 36 10 14 0 96 0 36 11 17 4 104 53 16 12 18 3 218 28 0 13 16 0 129 0 0 1<1 13 2 60 23 8 15 8 0 32 0 12 16 5 1 2 5 48 17 12 6 72 68 44 18 12 5 46 43 28 19 10 9 56 105 32 20 19 16 130 193 8 23 12 13 79 158 0 24 5 16 29 219 48 25 9 18 73 206 24 26 13 16 107 178 12 27 9 1<1 38 137 4 30 13 6 131 41 4 31 10 10 80 97 40 November 1 12 5 71 36 32 2 12 13 97 125 24 3 9 5 57 37 20 .,

" .. POOR SEED PRODUCTION IN TEPHROSIA VOGELII 9 son the flowers were not adequately pollinated, were not stimu lated to set, or were abscised prematurely. When buds and flowers at five stages were hand pollinated, treated with hormone, and obsel'ved for pods and seeds, receptiv .. ity of the stigma to pollen lasted for about 24 hours after an thesis. Immature buds 1 da~r before opening were not receptive to -. pollen. Mature buds ready to open also set poorly. The stigma ap peared to be most receptive to pollination at anthesis, and recep tivity continued until the flowers began to age. Day-to-day differ ences were insignificant in this experiment. (Table 4.) In another experiment hand pollination and hOl'mone treat ments were compared with controls. Unfortunately the tests were made when natural seed set was already high. Therefore the re sults do not show any gain in seed production due to hand pollina tion (table 5) and, 1n fact, suggest an adverse effect of the hor mone treatment on poc! and seed production. Artificial tripping may be accomplished by holding the flower rigid at the receptacle and depressing the wings and keel just in front of the point of the keel. With practice this may be accom plished in one movement with normal flowers. However, an esti • mated 20 to 25 percent of the flowers of P.I. 257533 cannot be tripped easily or cannot be tripped at all. Other lines behave simi '. larly. When the flower is tripped, the style is forced through the suture of the keel, usually carrying anthers and pollen along with it. The succulent epidermis of the stigma is bruised by this proc ess. Most stigmas so exposed are self-pollinated by the tripping motion. As pressure is removed from the wings and keel, the style withdraws, but pollen may be left behind. An anther or two may be caught in the suture of the keel and thus may be left outside the flower. The flower may be tripped repeatedly with less force but essentially similar behavior. A ra.re occurrence is the expul sion of a tiny cloud of pollen, which happens only the first time the flower is tripped. I n hundreds of tests flowers could not be

TABLE 4.-ihnO'ltnt of seeds 1)1'oduced from hand-pollinated, ho'r mone-tl'eatecl. Tephrosia buds and fiowen at different stages on consecuti'ue clays, .z .967 [5 pollinations per sample] Stage of bud or flow('r NoV'. 6 NoV'. 7 Nov. 8 Nov. 9 Mean I Number Number Number Number Nmnber • Immature bud 0 0 0 0 0 d M.ature bud 5 2 13 9 7.2 c .. Anthesis 30 22 32 22 26.5 a Mature flower 15 27 26 18 21.5 ab Aging flow('r 11 25 18 20 18.4 b Open-pollinated control 3 7 11 ,j 6.2 cd Mean 10.3 a 13.9 a 16.7 a 12.1 a

I In this and later tables, means of a set followed by same letter are not statistically difl'crcnt (p = 0.05). 10 TECHNICAL BULLETIN 1419, U.S. DEPT. OF AGRICULTURE

TABLE 5.-A1nounts of pods and seeds produced [1'om 25 Tephrosia ,.( jiowe'l's using va1''ious pollincLting techniques on 5 diffe'rent days, 1967

Technique Oct. 19 Oct. 20 Oct. 23 Oct. 24 Oct. 25 Mean Number Nmnbe1' Nmnbm' Nmnbe1' N1W!ber Number Pods Control 9 16 16 18 16 15.0 a ;.. Hormone 3 7 13 4 12 7.8 b Pollen 20 18 13 15 12 15.6 a Hormone and pollen 10 19 12 5 9 11.0b Mean 10.2b 15.0 a 13.2 ab 10.2b 12.2 ab Seeds Control 105 193 219 206 178 180 a Hormone 8 48 94 2 71 45b Pollen 188 226 160 160 123 171 a .... Hormone and pollen 79 29 73 107 38 65 b Mean 95 124 133 118 102

tripped artificially except when the receptacle was firmly held in the hand. Thus, T. 'vogeli'[ is self-fertile and self-pollinated. However, structural details of the flower often vary and impede self-polli nation. Some external enforcement of self-pollination seems nec essary for adequate seed production.

Role of Insects in Pollination Bees appear to have·a role in pollinating Teph1·osia. The flowers of T. vogeUi have a faint but definite, pleasant aroma and readily attract bees and moths during daylight. No attempt was made to monitor insect visits at night. Honey bees (Apis mellifem L.) were the most frequent visitors. They were too small and too .4 light to depress the flowel's. Two types of behavior were distin 1 guished, nectar seeking and pollen seeking. Nectar-seeking bees visited mostly the younger flowers. They landed on the wings, crawled to the receptacle, and presumably sampled from the nec .1- . taries of the receptacle. These bees usually did not touch the area ~ of the stigma. Pollen-seeking bees, on the other hand, visited -I chiefly the older flowers. They forced open the upper suture or .-\ short leg of the keel to remove pollen, but their behavior did not

appear to lead to pollination. ~i 1 A carpenter bee, Xylocopa b/,(lsUi((non£1l~ L., is sufficiently , large to accomplisll pollination. When it lands on a flower, the pet iole bends and the entire flower is depl'essed. The flower is then in a good position for self-pollination. Observations of hundr.eds of visits to flowers have shown that the stigma is rarely exposed by this bee. The carpenter bee searches for nectar and ordinarily does not gather pollen from Teph1'osia flowers. Nevertheless, bees carry from very small to very large amounts of pollen, Most of this pollen, when examined under the microscope, was from spe cies other than TepJlI·osia. POOR SEED PRODUCTION IN TEPHROSIA VOGELII 11

In one study plants were isolated in sCI'eened cages, Carpenter bees were captured daily and introduced to some cages (table 6). Although some flowers were self-pollinated and some pods and seeds were produced in insect-free cages, carpenter bees increased pod set twentyfold in cages where they were introduced, Insect activity in control plots also resulted in reasonable seed set. In ad dition, insects in cages or in the field increased the number of seeds . ;~ per pod over that of plants isolated from insects. In all experi mental plots, many more flowers were pollinated than pods were produced. Estimates of insect activity during the flowering season show ". some seasonal variation in .number of insects visiting the flowers. ... These differences are probably due in part to insecticide treat ments, dally weather variations, and seasonal variations in in sects present. Insect activity is highest in the early morning on dry, cool days. Each week during the 1966 flowering season examinations of .. hundreds of flowers abscised from their racemes revealed many small i'cars or scratches on every flower resulting from insect visits. Either honey or carpenter bees could have been responsible for such damage. Some of these flowers showed one or more an thers caught in the suture (fig. 1, B). Nevertheless, most of the flowers did not appeal' to have been tripped (style forced through keel). Many of these untripped flowers were self-pollinated. Thus, bees may facilitate self-pollination by changing the relative posi tions of keel, stigma, and pollen. The mild pumping action when the keel is de;Jressed may increase self-pollination even though the flower is not actually tripped. As tripping leads to self-polli nation, even the rare tripped flower may not be cross-pollinated. However, insects are not necessary for self-pollination. Flowers :~ opening in the laboratOl'Y, on inflorescences brought fl'om the field, were aHowed to dehisce and then examined for pollen 011 the "'. stigma. Self-pollination had occurred ill most·of them. Some self pollination and seed production occur even under insect-free con ditions in the field (table 6). A possible function of bees is to damage the stigma by breaking up the stigmatic epidermis. To test this supposition, a series of opening' buds was hand pollinated by two techniques. The stigmas of some buds were damaged with a pollinating needle before pol linating. Other buds were pollinated as gently as possible. The pollinated flowers were protected from further insect activity. Both pod and seed set were significantly higher following the .. damaging treatment (table 7) . Thus, the role of insects in self-pollination is clear. They may facilitate self-pollination, but apparently are not active in cross pollination of 'l'ephros£a. Insect activity is not sufficiently high at all times of the season. Although poor seed set apparently is the result of many interacting factors, the number of carpenter bees and their level of activity also afl'ect the amount of seed produced, especially when pollen production is adequate and weather favor able. ~ t-.o

TABLE 6.-Effects of introducing carpenter bees into cages of Tephrosia plants on pollination, pod set, and 8 seed production at 2 locations in Puerto Rico, 1968 lij @ Laboratory observations Field observatio.r.? Type of a~ structure Location Flowers Stigmas Flowers Seeds per observed pollinated observed Pods set pod ~ b:i Number Percent Number Number Percent Number Isabt~la. __ .. __ 31 20 8,448 70 0.8 6.6 8 Closed cage ,- ---- ~- - - . - - - ~- - - - . { MaYllgiiez. ~__ t-t 1,878 69 3.7 6.4 t;j Closed cage with introduced bees" Isabela _. ___ _ 65 68 3,040 532 17.4 9.4 tj Isabela ..• _ . __ ------10,808 1,159 10.8 9.7 Roof only . ~- - - . -. - . - - .•. - { Mayagiiez •... Z 1,880 429 22.8 8.2 I-' Isabela ...... 39 21 4,922 533 10.8 8.4 No structure - --~.-.. -- .... ---.-- { Mayagiiez ___ . 20 45 7,938 1,004 12.8 8.2 I-' ~""" c:l rn

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'" TABLE 7.-Pod and seed sei:. of T. vogeliias affected by damaged and 'Undamaged stigmas, 1967 [25 pollinations per sample] Pods set Seeds set Date Damaged Undamaged Damaged Undamaged stigma stigma stigma stigma Numbm' Number Number Number October 26 .... 18 14 132 70 27 16 14 76 0 30. 21 8 185 45 3L 17 11 99 62 Novembm' 1 .. 20 15 98 48

Mean - .. 18.4 (l 12.4 b 118 a 45 b

.. Abscission, Pod Drop, and Pod Shape , .. The unpollil1ated or unfertilized flower usually drops from the T. 1Jogeli'i, plant 30 to 48 hours after anthesis as the result of abs cission at the base of the pediceL The abscission process in other plants is believed to be controlled by growth-controlling sub stances produced by the developing fruit. The application of a small quantity of 1 percent indole-3-butyric acid in lanolin in hihits abscission of the T. vogelii flower. Treated flowers, whether fruitful or not, remain attached to the pedicel untH the end of the flowering season. . Unpollinated flowers occasionally develop parthenocarpic pods, which abscise 3 to 4 weeks after pollination. The production of parthenocarpic pods is a varietal trait, usually recognizable by '. the abundance of pods on the raceme. Parthenocarpic pods can also be identified by absence of developing seed when opened 7 or more days after anthesis or when held to the light 3 weeks after anthesis. Flowers that initiate pods remain on the plant but may be ab scised later, In early studies of pod drop, the abscission of par thenocarpic pods was confused with premature abscission of seeded pods. Because fertilized ovules are difficult to distinguish from unfertilized ones, distinguishing pods dropped due to failure of fertilization from those dropped due to other causes is virtu ally impossible. However, in varieties that do not produce typical parthenocarpic pods, some abscission of young pods does occur 2 to 7 days after anthesis. The phenomenon has occurred frequently enough after hand pollination under carefully controlled conditions to convince us that pods containing developing seeds often abscise prematurely. In three varieties the number of pods matured per flower was always much less than the number of pods set per flower, as meas ured 7 days after anthesis (table 8), In variety 10247 partheno carpic pods never occurred. Thus, a certain proportion of seeded

'...... H:>. TABLE S.-Fertility indices of :3 open-pollinated Tephrosia varieties of dib'e1'ent flowering seasons planted si?nultaneously, 1967 ~ I?:l 0 Pods set per Hower Pods mntur<...t per Hower Seeds per Rowe". Seeds per pod ~ Date Z 10247 6286 6285 102·17 6286 6285 102·17 6286 52':6 102·17 6286 6285 ..... ---~- .. 0 Number Number Number Numb.". N1I1nbc,. Number Number Number Number Number Number Number > Octobcr t-< b:j 'L .. ~ ....~ . ~ 0 0 0 7. .02 0 .02 11.0 c:::: 11. .02 0 .09 11.1 ~ 14 .06 .01 .15 8.9 I?:l 18. .06 0.04 .03 0.02 .32 0.06 9.0 8.0 21 .06 .02 0 .05 .02 0 .42 .23 0 8.8 9.9 ~ 24 .07 .04 .02 0 .01 .01 .05 .08 .09 10.2 6.6 9.0 Z 28 .10 .04 ~ .04 .05 .03 0 .29 .31 .01 9.2 8.2 7.0 ~ . Novctnbcr ~ 1 .17 .17 .11 .08 0 .03 .70 .02 .27 9.0 5.0 6.2 ~CD 4 .16 .16 .48 .09 .08 .02 .69 .10 .14 7.3 8.4 6.5 B .14 .14 .33 .10 .09 .01 1.05 .58 .52 7.4 7.6 7.1 c:::: n. .18 .37 .05 0 .26 0 6.2 fn 15 .32 .25 .17 .04 .86 .67 7.1 -'6'.9 ... '=' 18 .. .42 .09 1.15 6.1 ..,I?:l 22. .30 .02 .50 5.0 25 .39 .03 .51 5.0 ~ Mean. '''' ...... " .08 .12 .22 .04 .05 .02 .36 .33 .35 9.2 7.4 6.5 ~ > ~ ~ 0 c:::: Ei c:::: E3

~ .. j. ..~_.. ~ ~ l. ___ !,_..;____." ~ ~ ~" .... ~. ~., )~""'""'."~'_'"''''''''''.'.C'!:"O,-•. ~"~~ ,,,-,,. •._,._ ••_._.1t_.,•._ ._ .•~~....• ..._. iL. _.•Ii: ...... ! ~.1.....3.· .• i._•.•. .••._t..... j,; ._ ••.. POOR SEED PRODUCTION IN TEPHROSIA VOGELII 15

pods is lost by abscission. As judged from collection of abscised pods from the ground below the plant and by shaking the inflo rescence, abscission seldom occurs after well-seeded pods are 5 or more cm. in length. .. The shape of the seed pod bears an interesting relationship to the number of seeds. When only a few seeds are present, they are invariably near the stigmatic end of the pod. The mature pod then assumes a falcate shape (fig. 3), in which only the seedy part is swollen. This phenomenon suggests that ovules are fertil ized in order as pollen tubes enter the ovary. Occasional gaps in the seed pod show that this ordered arrangement may sometimes break down. When the seeds per pod were counted for a large number of ... pods, the data were distributed nearly normally, but with a skew ing toward excess pods with few seeds (fig. 4). The mean number of seeds per pod was 9.6 compared to lOA available ovules. When seeds were assigned position numbers counting from the stig matic end of the pod, the mean position was 7.8. This also reflects .. the tendency for ovules near the stigmatic end to be fertilized .. first. .. Effects of Hormone Treatments Various types of hormone applications have been used in con junction with different pollination treatments in attempts to im .. prove pod and seed set of 1'. 'Vogeli-i. The systemic growth regula

. A

• •

llN-35495 . ... FIGURE: a.-Left, T. vogelii pods of various shapes as affected by number of seedSjright, their seed contents.

.. 16 TECHNICAL BULLETIN 1419, U.S. ,DEPT. OF AGRICULTURE

60 ! ~ i i ,~~ 1 ... 1 I 40 V'I o o 'Q...

o o 5 10 15 20 SE E0 S PER PO 0

FIGURE 4.-Distribution of pods by number of seeds per pod in T. vogelii.

tor N-metatolylphthalamic acid applied to entire plants in aqueous sprays of 500, 1,000, and 2,000 p.p.m. did not affect pod set. Parachlorophenoxyacetic acid (peA) and indole":3-butyric acid CIBA), when applied in lanolin to the base of the flowers at , rates of 0.2 and 0.8 percent, respectively, appeared to increase pod j ..) set (table 9). However, since the pods were usually seedless, the i ... 1 1 TABLE 9.-Effects of hO?'tnone treatments on pod set of emascu ,.; lated, cross-pollinated, bagged flowers of T. vogelii 1 J Hormone Flowers I treatments • trf:llted Pods set Seeds per pod ... I Nltmber Percent Number -cl None (check) 160 13.1 5.0 i Lanolin 95 12.6 3.7 i ,peA 101 70.a 1.0 IBA .3 97 78.4 i peA + IBA 99 60.6 .7 ..(;

1 Data taken from unpublished reports of Federal Experiment Station, Puerto Rico, 1955. No statistical analysis was possible. • peA is parachlorophenoxyacetic acid; IBA is indole-a-butyric acid. j .- t I 1 ,,", '. POOR SEED PRODUCTION IN TEPHROSIA VOGELII 17 treatments were ineffective in increasing seed production. In later experiments treatment with IBA increased seed set at the begin ning but not during the last part of the season (table 3). The effect of hormones is apparently to delay abscission. There fore hormone treatment would be expected to be most valuable when pods were abscising prematurely. This probably explains why hormones increased pod and seed set in some but not other experiments. When abscission is not occurring, treatment with hormones is not likely to increase pod and seed set. -- POD AND SEED YIELDS Factors Affecting Seed Yield Seed-yield differences of Tephrosia vogelii are caused by many factors other than the partial sterility of the species. For exam ple, time of planting affects maturity, and maturity interacts with day length to determine season and duration of bloom. Fertility of the soil, pH requirements, weed competition, and other agro • nomic factors affect the size of the plant and thus the number of flowers produced. Seed production could be increased by appro .. priate agronomic treatments alone simply by increasing the num ber of available flowers. However, the problem of poor seed set is a matter of poor production per flower. Consequently, three in dices of production have been used: Pods set per flower, pods ma ttn'ed per flower, and seeds per flower. In addition, the number of seeds per pod was investigated in some instances. Effects of sea son, variety, weather, and location have been evaluated over a 4 L year period in terms of these indices. All varieties apparently show a short period of sterility when flowering commences. This initial sterility, involving' the first flowers, is not changed by planting date or other factors. Hand pollinations during this period are not usually successful (table 10). Pollen germinates successfully in the style, however, during this period. Thus, failure of pud set is not due to poor pollen pro duction 01' fertility. Hormone treatments after hand rollination were found to restore normal pod set (table 3). This suggests that sterility of the first flowers is due to a failure of the hor mone-controlled pod-setting mechanism. As few flowers are in volved, this temporary sterility is of minor importance. All varieties also show an end-of-season sterility involving the final buds of each raceme. These buds and flowers are often un • derdeveloped and show signs of nutritional deficiency. Adequate care of the plants, especially near the end of the flowering season, • might dela! the onset of terminal sterility. When distinct varieties are planted at the same time, they ma ture -at different rates and bloom at slightly different seasons. Comparisons of three such varieties showed differences in fertility indices (table 8). However, when these varieties were compared .. over periods of time when all were blooming, the variation in fertility indices due to dates was more pronounced than the .. 18 TECHNICAL BU;LLETIN 1419, U.S. DEPT. OF AGRICULTURE >. TABLE 10.-Seasonal changes ,in pod set of T. vog-elii XP.I. 257588) w'it/Lout and wUh hand pollination of buds and fiowers, 1967

Unpollinated controls Hand pollinated Date of pollination Flowers observed Pods set Pollinations Pods set Number Percent N1tmber Percent October 6 ". - .... -> - .. - •• .... -----_ .. 39 0 35 0 13 .. -. . - 51 0 47 2.1 20 - ... ,.. ... _.... - 58 12.IJ 43 11.6 27 -..,. .. -----_ ... _.... -. -- ... - 52 23.1 50 28.0 November

- 50 18.0 53 32.2 3 ... -.. - . - .. -.. -.. -- ..... -----~ 22.8 59 62.8 10 - .... -~--~- ... --. -- ..... - 57 17 ------50 30.0 50 66.0 23 ...... "'------50 42.0 52 54.0 _.. 34.8 Mean " ..... --~----- 18.9 variation due to varieties (table 11). These data suggest that weather conditions affect several varieties similarly. • When a single variety was planted in several locations in Puerto Rico, fertility .indices as well as total seed production dif fered with locality (table 12). Fertility was highest in the cool, wet climate of the mountains (Castaiier) and lowest in the hot, dry valley (Lajas). Site of planting affected not only the number of pods set but also the number of seeds per pod. The weather data and climatic characteristics of four planting sites are shown in table 13. High humidity, rather than altitude, rainfall, or tem ,xl perature, is probably the most important factor affecting fertility.

Effects of Weather Changes Although differences in fertility of T. vogelii from one location to another may be demonstrated within anyone locality, day-to day differences may be much greater (table 3). Of the variables affecting flowering and pod set--w,eather, day length, soils-only the weather changes considerably on a day-to-day basis. The pos sible detrimental effects of rainfall on pod set were first suggested in 1955. However, sufficient data were not collected to derive cor relation coefficients. Attempts to correlate weather variables with pod set of flowers opening on the same day have not been successful. The reason for failure is evident: The life of the bud and flower extends for about 2 weeks and includes meiosis and pollen production, polli nation and fertilization, inhibition of abscission, and stimulation of pod development. Weather variables may affect pod set at sev eral phases of the flowering and fruiting process. The effects of the following 10 daily weather variables on pod and seed set were determined in one experiment throughout the .. flowering season: Rainfall, highest temperatur.e, hours over 90° '- +"l''''~".....'-,. 1,:""""" ,. r 'j~ ~'''~1J j;' ", "T ,~'" '. "t' ".. ~~",i ., , .-'.\' L 1 '" f .. r"~'

o'"d TABLE ll.-Pertility indices during /lowering of 3 Tephrosia varieties, 1966 ~ '~ Pod. set per flower Pod. matured per flower Seeds per flower e5~" Date 10247 6286 6286 Mean 10247 6286 6285 Mean 10247 6286 6285 Mean Number Number Number Number Number Number Number Number Number Number Number Number October 0.02 a 0.42 0.23 0 iJ:22 e 21 _ •• __ .... T ______.. ___ O.OG 0.02 0 0.03 e 0.05 0.02 0 ~ .02 JJ3e 0 .01 .01 .01a .05 .08 .09 .07 d a 24 , .••.••• _.•.• _•. _ .04 .04 .01 .20ed 28 _•. _. ______.••••. .10 .04 .04 .06 e .05 .03 0 .03 a .29 .31 ~ November o 1 .. ,._. ______... _. .15 b .08 0 .03 .04 a .70 .02 .27 .33 be .17 .17 .il .14 .41b z 4 -._ •.. __ ••. ____ .16 .48 .27 Ii .09 .08 .02 .062. .69 .40 I-< ...• .16 1.05 .58 .52 .72 a 8 .14 .14 .33 .20 ab .10 .09 .01 .07 a Z -~------~~ .16b .32 ~ Mean,. .095 a .112 a .165 a .124 .062 a .038 a .012 a .037 .55 a .24 b l':I ~

>~ ~

~ I-<

... ~ 20 TECHNICAL BULLETIN 1419, U.S. DEPT. OF AGRICULTPRE

TABLE 12.-Fertility indices of T. vogelii at 4. locations in P1terto R-ico, 1967 Location Pods per Seeds per Seeds per flower flower pod Number Number Number Castaner' ~·~_~ ___ T~ __ ~~_~ _____ 0.15 ab 2.2 a 14.4 a

Castaner -~ .. - .... -._ .... ---- .14 ab 2.0 a 14.5 a Las Mesas ~ -- ~ - .. ~ ... - "" ~ - ~ .11 be 1.0b 9.0b ., Mayagliez --- ... - ....,,. .... ~~""-~~ .18 a 1.8 a 9.6b i Lajas ,., - . - _.. ~ - -- .06 c .6b 9.5b , This plot was limed to correct acidity of soil.

TABLE 13.-Sonte vUl1'iables assoC'iated with 4- 1)lanting gites of T. vogelii in Pue1·to Rico, 1967 Mean Mean Rainfall high low General Location Altitude during tempera- tempera climatic flowering ture l;ure characteristics Feet Inches of. of. Castaner 2,200 8.3 83.7 60.4 Cool, wet; in mountains. Las Mesas 500 Hot, moist; in foothills. Mayagliez 35 9.1 93.9 74.8 Hot, moist; on coast. Lajas 25 '11.2 89.1 67.7 Hot, dry; in valley. 'Includes 8.7 inches of rain that fell during final days of flowering when hurricane passed nearby. .. F., hours over 80°, hours under 75°, lowest temperature, highest relative humidity, hours over 90 percent relative humidity, hours over 80 percent relative humidity, and lowest .relative humidity. Measurements were begun 10 days before flowering and termi nated 8 days after. Each day buds beginning to open were identi fied with dated tags. The plants were observed several weeks later for pod set. Correlation coefficients between pod and seed number and each of the 10 weather variables were then calcu lated for each day from 10 days before to 8 days after flowering. The coefficients were then gI.'aphed by day to determine the effect of each variable on pod and seed set over the 19 days. Correlation coefficients were moderate to low, as would be ex pected when many factors are interacting. Most of the coefficients are not in themselves significant. But correlations in one direction for several consecuti ve days suggest definite effects of weather variables on pollen production and pod and seed set. Daily rainfall was not strongly conelated with pod or seed set before or during anthesis (fig. 5). Rainfall after anthesis may have had slightly beneficial effects on the retention of pollinated flowers. This could have been a spurious correlation due to the ef fect of rain 011 temperature. Both low and high temperatures were apparently negatively correlated with pod and seed set before but not during anthesis (fig. 6). Low temperatures were po~itively correlated with pod and seed set 2 to 4 days after anthesis. , ;L ,I' ""':0-' "~,.-. ~ ""","t"" -". ,,... ~Y,,~"""""" .. >-'-.>- <:;:,,~,' "~ "r" r ' "',l" ~ ,l ~- ,' ,., ,:=') 1. f j.. if ~ 1 • .;' • '" •

0.75 ~ CORRfLATIONWITH RAINFAll: ~ trJ Pod set t:l ~ 0.50 I- ••••• 11 •• Seed set ~ 0 ~ § :z 0 ~ ...., u 0.25 .... u- 0 L,;,~ Z ~ .... 0 u Z 1-3 Z 0 trJ 0 ~ ~ ::t -<~ ~ ~ 0 0::: 00. c.:: -0.25 .... 0 > u -< OF 0 DAY Cl -0.50 r FlOWERING trJ ~....

-0.7 5 -10 -5 0 5 10 DAYSBEFORE AND AFTER flOWERING l'\:) FIGURE 5.-Correlation of pod and seed set of T. vogelii with daily rainfall from 10 days before to 8 dayS after anthesis...... tI:) tI:) 0.75 POD SET CORRELATEDWITH: ~ t:rJ 0 Highest temperature ~ 0.50 Z --- Lowest temperature ,.... 0 :z~ ••••••••• Hours over 80° F. u..I ~ U 0.25 b:I u... c::: u... ~ u..I o f;;j 1-3 u I-C z o Z o ..... ~ ~ c( ..... -' •• ~co ...... '.-. ,-----.. ~ -0.25 .-~.. /. c:: c.:: /, -- o ',/ --- rn u ./ ~--- .....~ ...... DAY OF ~ -0.50 _./ '"d flOWERING .~ ~ > L-' ---I'---L....-~--'----'--.l----L.--...... IL-...... 1.....---II-....L.-L_-L-...... L_..L..---L_L-...L.---'_...J -0.75 ~ I-C -10 -5 0 o 5 10 c::: ~ DAYS BEFOREAND AFTERFLOWERING 1-3 .~ ~

.. r- I _~_.~__.,___ ~ ~ .. ~ . I J ~ .. ,.. & ""H... "v.-n~_~~. "'....~"<'i~O_:...,ik",->{;--,"'-::;')'..' '1"7f'r" ~'''~....'.''" ""y""""y'>'~·,r"'"'i"~"''',~'''''!·f. a "",~ 110' " . .I' \0- ~ ,~'.

~ 0.75 ~ SEED SET CORRElATEDWITH: 00 l:"J Highest temperature ~ 0.50 I --- lowest temperature ~ t- 0 :z ...... Hours over 80° F. •••.'.•• § IoU .,,.. u .. C 0.25 >-3 u.. ••• ~ u.. \ ' "'..1\, 0 IoU /~.' . 0 Z \/ .. 1-'4 U ...... -- Z :z 0 .-. 0 '~ t- ~ -.I •• ,.' .. II: IoU •• ~.'...... " J ~ < ~ -0.25 t·· )"--- ~ / DAY OF > u ..... ,,.----~ < ..... Oy/ flOWERING 0 -0.50 I ~ ~ -0.75 IL-L----1L-L---1--L--L--1_L-..L-+..-L---'.-L--L--L---1----L-L-...I...--: 10 -10 -5 o 5 DAYSBEFORE AND AFtER FLOWERING

FIGURE 6.-Correlation of pod and seed set of T. l10gelii with three temperature measurements from 10 days before to 8 days ...,;, after anthesis. CI:I 24 TECHNICAL BULLETIN 1419, U.S. DEPT. OF.AGRICULTURE

High humidity measured by several indices was positively cor related with pod and seed set during preanthesis but not during anthesis (fig. 7). Low humidity was correlated with pod and seed set 2 to 4 days after anthesis. These effects of weather variables are summarized in table 14. Each variable is expl'essed on a scale of - 3 to +3. Pod set and seed set were highly correlated; therefore weather variables af fected these indices similarly. All conelation coefficients measured must be interpreted with caution. Since they wel'e low to moderate, no single weather vari able nor time period can be selected as the most effective with re spect to pod and seed production. Nevertheless, the trends in corre lation coefficients suggest that weather affects physiological pro cesses of the plant. Before anthesis, which is during pollenproduc tion, moderate temperatures and high humidities appear to be de sirable. During anthesis, pollination and fertilization appear to be slightly resistant to external influences. After anthesis, rainfall, moderate temperatures, and low humidity-a contradictory com bination of circumstances-appear to be necessary for several days until the pod is firmly set.

Correlation of Climatic Variables With Pollen Production and Self-Pollination The number of T. vogeli·i flowers with unopened anthers and the number of self-pollinated flowers were noted daily, The data were compared with weather variables by correlation coefficients. Naturally weather variables could affect pollen production and self-pollination only during preanthesis. The correlation graphs for the 10 days preceding anthesis suggest that this period may be divided into two or possibly three subperiods (fig. 8). During the first subperiod from 7 to 10 days before anthesis, rainfall was not correlated with the failure of anthers to dehisce

TABLE 14.-EjJects 0/ 10 weather v(//riables on pod and seed set 0/ T. vogelii beloTe, cl7t'1'ing, and a/te-.,. anthesis, 1967 1 Period in relation to anthesis Weather variable Before During After

Rainfall ~ +1 0 +2 Temperature (0 F.) : Highest -1 0 0 ... Hours over 90 -1 +1 +1 Hours over 80 -1 0 +1 Hours under 75 -2 0 +1 c Lowest -3 0 0 Relative humidity (percent): Righ('st +3 0 -1 Hours over 90 +3 0 0 Hours over 80 0 +1 0 Lowest -1 0 +2

I Expressed positively and negatively on a scale of 0-3. i' POOR SEED PRODUCTION IN TEPHROSIA VOGELII 25

or later self-pollination. High temperatUl"es and ,high humidities were negatively correlated with non dehiscence of anthers and positively correlated with self-pollination. During the second sub period fl·om 3 to 6 days before anthesis, l"ainfall had little or no effect, and high temperatures were positively correlated with non dehiscence of anthers. These effects were reversed again during a possible third period from 1 to 2 days before anthesis. .. The breakdown of preanthesis into subperiods emphasizes that several distinct physiological processes occur that may have their own optima of temperature and humidity. They include meiosis, pollen nutrition, and maturation of the anther. Whether the an ther dehisces or self-p01ljnation occurs depends on each preceding event. Other factors, such as failure of pollen to germinate, also contribute to pod- and seed-set failure. This is probably the rea ' ... son that the curves showing the relationship of weather variables to anther and pollination failure do not perfectly parallel the curves showing the relationship of weather variables to pod and seed set. The relatively low but significant negative conelations between the two sets of variables, pod and seed set versus failure of an .. thers to dehisce (table 15), also suggest that other factors have been overlooked. The relatively high correlations within the'two sets of data suggest that pod and seed set are similar measures of the same phenomenon, fertility. However, nondehiscence of an thers and amount of self-pollination are correlated but do not measure the same phenomenon.

Effects of Modified Euvironments To test the effects of weather variables on pod and seed set of T. 'vogelii, three modified environments were devised and their ef fects on pod and seed set were compared with those of the natural environment. Each type of environment was replicated four times in small plots of 144 square feet. One type consisted of plots shel tered from rain with a polyethylene-covered house 6 to 8 feet tall. The plastic sides of the house reached to within 3 feet of the ground. The plants were watered with an overhead sprinkler for 1 hour three times a week. The second type consisted of the same kind of sheltered plots, but soil moisture was maintained by peri-

TABLE 15.-Correlation coefficients among amoun.t of pod and seed set of T. vogelii and amount of anthe?· failu?·e and self 1Jollination, 1967 Failure of Variable Seeds set anthers to Self dehisce pollination

Pods set t 0.98 Z -0.30 0.03 Seeds set Z _ .27 .03 Anther failure t -.81

• t Highly significant (p <0.01). Z Significant (p <0.05). ,26 TECHNICAL BULLlETIN 1419, U;S. DEPT. OF,AGRICULTURE

~, ~, • e•• ) 7. ,~ .. ,. ~. \ ...~. ) .. / .. ' . "0 /' .•. ,/ ••~ / i ", ....' ,...... •• '"', ...... " .

',- I ·• I, I ·• I ·• · o V'I o V'I o V'I -...... V'I N o V'I ...... o o ,0 o o IN31)1~HO) NOIIVH~UJO) ~;r,:li'?;r"~';"''''''''i. ~.'..j..."':\r-<'~~~i--!'.J!~'r:.v:;~;J,-C,:. _.,-;'<~';r"v

loti 0.75 r' ------...------ ~

0.50 ~ I, loti I / , ~ Z / g w , C '-' 0.25 ~ \4. \4. ••••• ...... - '"' "\... .•• ~.-' A - " W ...... -:\,... ~ o .. .. u ...... ,/ .. . Z o..I:.·········..• ••••• /' -...... z ... 1/ .... ••• ------o "" I- " ...... "', ,--, .•...•• .•.... ~ <.....I I:II .•...... ------~ ~ -0.25 ...... ,."~/ -...... ~ ex: ,/ o u SEEDSET CORRElATEDWITH: DAY OF > Highest humidity flOWERING < -0.50 -- --- Lowest humidity ~ ••••••••• Hours over 80% R.H. E::

-0.75 LI -----'L--L-.-l._L--L-l~_L_-1.._.L.-+_.L__L___..L_.L___'___I_....L._____'L._.a.._ - -10 -5 o 5 10 DAYSBEFORE AND AFTERFLOWERING

FIGURE 7.-Col·relation of pod and seed set of T. vogelii with three humidity measurements from 10 days before to 8 days after t..:l anthesis. -::J 28 TECHNICAL BULLETIN 1419, U.S. DEPT. OF AGRICULTURE odic irrigation with a garden hose. In the third type, unsheltered plots were watered from overhead on the same schedule as were the sheltered plots. In the fourth type, unshelter.ed plots received only natural rain or irrigation by a garden hose as necessary. Treatments were begun 2 weeks before flowering. During the short (3 weeks) but intense flowering season, pod

RAINFAll . HIGHEST TEMPERATURE HOURS OVER 90· ~

..;0:5

~\O ':OAY:S '.0 ;IO:!iUS' ",0 ;\0 :OA'YS (0 ~------~ .~------~ HIGHEST RelATIVE HUMIDITY

J.j \

-I

t..' -10 o ·10 O'AYS o - flOW.ERS .WITH :NONDEHISCENT 'ANtHEMS ·--SE[F~POLUNATEP HOWEllS

FIGURE S.-Correlation of weather variables with nondehiscence of anthers and self-pollination in flowers of T. vogelii from 10 days h'i!fore anthesis to anthesis. \..i . ! POOR SEED PRODUCTION IN TEPHROSIA VOGELII 29

and seed set were recorded for all flowers that opened on labeled inflorescences. ·Weather variables were measured with a contin uous strip l'ecorder in each modified environment. During the 3-week flowering period, both pods pel' flower and seeds per flower significantly increased (table 16). These differ ences were particularly Im'ge between the second and third week of flowering. However, the modified environments did not affect the seed set nor were there significant interactions of enviTon ments and dates Oil pod and seed set. Neither environments nor dates significantly affected seeds pel' pod (data not included here). The rainy season terminated rather abruptly the week before flowering and little rain fell during the tesh; (table 17). It did not touch the sheltered plants. Temperature conditions, recorded as 0 high, low, hours over 90" F., and hours over 80 , were remark ably stable. with minor day-to-day fluctuations. Moreover, fluctua tions with time were no Im'ger than fluctuations due to the modi fied en\'ironments, and no warming or cooling trends could be dis eerned d u ri ng the 5 v.. ·eeks that records were taken. Daytime tem peratures under the pla~tie ~helters were generaI1y a few degrees higher than the outside temperatures, but nighttime tempera tures wel'e sim.ilar in all environments. Relative humidity, recorded a~ hours over 90 percent and mean low, fluctuated widely with temperature. Humidity of 90 percent or over OCt'lIlTed en'r,)' night. Mean low humidity ,,'as lower inside than outside and decreased with the flowering season. The in crea~e in pod and seed set over the observation period thus cannot be related either to changes in the weather or to induced environ mental modifications. A possible cause for increased pod and seed set is that insect activity increased during the flowering period. This trelld was observed but not measured. Another possibmty is that the trench; are only a reflection of the normal change from early-season ~terility to fertility. However, in none of the envi ronments did pod and seed set approximate the desired level. None of the experimental \'arinbles showed any triggering effect • on fertility or sterility.

Varietal and Spcde!:i COJuparisolls Seeds of T. t10gclii have been~'eceived from sources as distant - as Liberia and Madagascar. Of some 40 introductions, none have set pods and seeds freely. Nevertheless, some differences in total seed production ha\'e been observed among introductions. These result in part from differences in number of flowers or season of flowering. Accurate measurements of such differences are. only meaningful when varieties flower simultaneously and when pod and seed set are recorded on a per-flower basis. Only a fe,,, data of this type are availahle (see tnble 10). The variety llsed for most of the studies reported here-Fed em! Experiment Station 10247 (P.I. 257533) from Panama, al though probably not nati\'e there-was selected because it pro duces more seed than other \'arieties. However, pod and seed set in CI-' TABLE 16.-Effects of 3 modified and 1 natural environment on 1}od and seed set per /lower during 3-week o /lowering season of T. vogelii, 1967 Sheltered plots with- Unsheltered plots with- t;ij Mean o Week Sprinkling No sprinkling Sprinkling No sprinkling II: Pods Seeds Pods Seeds Pods Seeds Pods Seeds Pods Seeds ~ Number Nu.mber Number Number Number Number Number Num.ber Number Number 1 0.09 0.88 0.10 0.86 0.05 0.41 0.10 0.91 0.08 b O.76b ~ 2 .. ~.. _._ ... , .. ~. .12 .80 .11 .88 .09 .73 .12 .79 .11 b .80b ttl

3 . ., ...... ~ ... , - .. .34 1.10 .29 1.50 .24 .95 .25 1.20 .27 a 1.18 a Mean ...... 18 .92 .17 1.08 .12 .70 .16 .96 .16 .91 ~ Z ~ iI>o ..... 'fABLE 17.-Mean weather conditions outside and inside shelte1's before, du?ing, and after T. vogelii /lowering, ~ 1967 C! Week during flowering rn Before After flowering Weather variable flowering, 1st 2d 3d outside ~ Outside Inside Outside Inside Outside Inside Outside Inside ~

Rainfall (in.) .. . ___ .... _. ..' 4.24 0 0 0.21 0 0.27 0 0.40 0 ~ Temperature (0 F.): >- High '" .... _•.• _...... 91.7 93.0 95.9 93.1 93.5 93.4 94.4 91.4 94.1 Low ...• _..•..• _...... _ . _ •.. 72.0 73.6 72.5 70.6 71.2 71.4 69.7 71.9 71.0 Hours over 90 .. , ...... 3.1 3.4 6.8 3.0 5.2 4.0 4.6 2.9 4.0 eo Hours over 80 ...... - ... ~ .. ~ 9.4 9.3 11.9 9.1 10.3 10.0 10.9 10.7 9.9 Relative humidity (percent): 8 Hours over 90 .. ___ ... _.. __ _ 7.4 7.0 7.6 8.0 7.3 6.8 7.3 7.6 7.0 Low ... _..... _____ . __ . ___ .. __ 52 50 45 46 43 44 42 47 43 ~

t _ r ...... t,M.....='~._...·,,,_,~__..~__.t. __ ,l. __ ..r~._.£~___.i.••._~'l.....-~-.~.' ~••,., •.•_ ...... !_''M!:.-..-'"'---...... ;.,."',- ...... ,.) ..,...... _..- ..- .. ' - ."""---....-. ..' .....""....' '. POOR SEED PRODUCTION IN TEPHROSIA VOGELII 31 this val'iety have never approached an acceptable level. Several selections-6273 bulk, 62121 bulk, 6286 (36-2), 6285 bulk, 6273 (31-6), 6286 (36-4), 6286-observed for the characteristics of sterility found in 10247 demonstrated the same problems, includ ing pollen abortion and failure of anthers to dehisce. The selec tions differed in amount of parthenocarpic pods produced. How ever, comparisons were not sufficiently extensive to rule out the possibility that sterility mechanisms are stronger in some intro ductions than others. The following- TCjJh /'o,<;;a species wel'e examined while flower ing and fruiting: T. candida, (Roxb.) DC., T. eh'renbergiana Schweinf., T. heclmw.l/llial/(l Harms, T.illcwl/a Grah., T. lepida Bak. f., T. PII/'PW'C(I (L.) Pel's., T. sina}JOIt (Buc'hoz) A. Chev., T. slIiJtl'ijiora Hochst., T. vestita Vog., and T. villosa (L.) Pers. The most striking characteristic of these species was their heavy pod and seed set. Most of the pods were completely filled with seed. Each species showed evidence of automatic self-pollination, in cluding dehiscence of anthel's before anthesis. All but one of these species are small shrubs with dull flowers. T. C(ll/dida resembles T. vogelii. in that the flowers are large, white, attractive, and al'omatic. Furthermore, the flowers of this species al'e visited by bees. T. candida differs from T. vogelii in that from one to six flowers are borne at each flowering node. Not all the buds at a node come into bloom, probably because of com petition for nutrients. It is therefore difficult to determine the number of flowers that have actually bloomed in old inflores cences of T. candida. This. in turn, makes exact counts of pods and seeds per flower impossible, unless individual flowers are tagged. However, judging from flower abscission in this species, only a small pel'centage of flowers falls withr)l1t setting pods. At anthesis the stigma is normally covered with pollen automatically. Pollen abortion was low in several dozen flowers examined. Thus, the sterility mechanism of T. 1logeli'i is not operating in T. candida. • DISCUSSION Evolutionary Status of Tephrosia 'vogelii T. l'ogelii cannot be considered either a cultivated or a wild spe cies. Its use as a dooryard plant for poisoning fish, a common practice in primitive societies, involves a certain amount of con Ir scious control by man. Seeds must be gathered and stored. Plant ings must be made. Yet the plant also can be found in the wild state, where it is preserved by man as a desirable intruder. Information is not available on the capacity of T. vogelii to sur vive in the absence of man's activity, but we have seldom observed it to be weedy. For example, in Puerto Rico and the Southern United States it does not regro'" after severe cutting and mowing; the seeds do not remain for long periods in the soil to give rise to weeds; and it does not invade or colonize disturbed areas, These '1 1 l 32 TECHNICAL BULLETIN 1419, U.S. DEPT. OF AGRICULTURE ..\ J characteristics of the species are useful in understanding the sig nificance of partial sterility. Probably T. vogelii should be considered as semicultivated, with a potential for evolving either as a cultivated or as a wild plant. This in-between state of imperfect adaptation may be the principal cause of the poor seed set. The fixation of deleterious genes by inbreeding in small populations may have caused insta bility of certain reproductive processes, as well as the rigid self pollination now characteristic of the species. Whether 01' not T. vogelii is a partially sterile species is in part a matter of definition. The survival of any species suggests that its reproductive processes are adequate. A certain amount of re productive 'vastage can be considered normal. The amount varies among plant and animal species. Thus, seed set of T. vogeUi is poor only when viewed from an agronomic viewpoint. Neverthe less, when man directs the evolution of a species, he sets .his own goals of what is adequate or inadequate behavior. The variation among accessions of TepiLrosia in seed set, as well as in other agronomic characteristics, suggests that cross-breeding proce dures would be useful in increasing seed production. Certainly in any plant-breeding program seed production should be given a high priority.

Causes of Poor Seed Production Of the various factors that could cause poor pod and seed set in T. vO[felii, the most evident is pollen abortion. When pollen is not viable, pod set cannot occur r~gardless of later events. Cross-pol lination of a particular flower to compensate for its pollen failure is virtually impossible. Although visible signs of abortion can be seen soon after meiosis, consistent positive correlations of pod set with high humidity during the preanthesis period suggest that the visible abortion is but one sign of maldevelopment of pollen. Weakness and inviability of morphologically sound pollen grains probably also contribute to partial sterility. The role of insects in pollination seems to be clear. Large bees cause redistribution of the pollen without rupturing the keel, eaJ'l'ying pollen, or cross-pollinating. \Yhether or not bees damage the stigmatic epidermis, thus preparing it to receive pollen, is - questionable, for the gentlest hand pollination, as well as self-pol lination, results in adequate pollen germination. It is not certain whether varieties dUrer ill this respect, but insect activity is not always sufficient to self-pollinate all flowers. Lack of pollinating insects could be the predominant cause of poor pod and seed set when pollen production is adequate. Postfertilization effects on POOl' seed production are probably associated with premature abscission of the flower. That abscis sion occurs cannot be doubted. Between the time necessary for a pod to set and to mature, a variable but significant number of pod::; is always lost. Although adequate experimental data are not POOR SEED PRODUCTION IN TEPHROSIA VOGELII 33

available, eal'ly pod abortion is thought to be caused by adverse weather conditions, especially temperature extremes, during the first few clays after flowering. The effect of hormone treatment on nod set is to delay the abscission of the flower or pod. This per mits the pod to develop normally if fertilization has occurred, or it may stimulate pal'thenocarpic pod set. Minor causes of poor pod and seed set are flower abnormalities including short stamens, weevil damage to flowers and pods, and perhaps nutritional deficiencies. Since many factors are involved, it will often be difficult in spe cific cases to pinpoint 01' identify the causes of poor seed produc tion .

.Recommendations for Seed Production and Further Study To maximize seed production of T. vogelii, several distinct fac tors must be coordinated. First, plantings must be made in an ap propriate environment. A moderate temperature and regular light rainfall probably would he desirable. These conditions are found at the higher altitudes in Puerto Rico. Second, plantings '... shouJd be made as early in the year as possible and given the best care to insure the largest and most vigorous plants possible. To insure the presence and activity of pollinat1lig insects, planting close to weedy areas and in ahandonedfields where dead trees or legume plantings occur might he advisable. The plants will need special attention during the dry season when flowering begins so that neithe1' soil moisture nor nutrient status of the soil limits growth. Mild insecticides applied during the afternoon can be used to control weevils and aphids. Further study of the seed-production problem should follow five directions. First, existing varieties of T. vogelii should be tested over a wider range of environment. The range in Puerto Rico may not suffice to demonstrate adequately the seed-setting po tential of the species. The possibility of varietal-environmental interactions should he i.nvestigated. Second, the use of chemical compounds in preventing abscission should be studied. Acceptable compounds must be ~conomical and applicable by spray tech niques. Third, increasing seed yields through breeding must be considered. An initial step in such a program could be the selec tion in F /s for high seed yield per plant. The amount of rotenone production, although probably of more importance than seed production, could be measured after screening for seediness. The heritability of seerl yield should be determined. Fourth, the effects of cultural factors on seed production, including nutrient status and insect-control practices, should be studied. Finally, methods are needed to increase natural populations of carpenter bees. These suggestions depend on whether T. vogelii becomes a crop plant. Extensive breeding work and testing in diverse environ ments will not he justified unless the prospects are favorable for producing rotenone economically from this species. 34 TECHNICAL BULLETIN 1419, U.S. DEPT. OF AGRICULTURE

LITERATURE :CITED (1) BARNES, D. K., FREYRE, R. E.., HIGGINS, J. J., and MARTIN, J. A. 1967. ROTENOID CONTENT AND GROWTH CHARACTERISTICS OF TEPHROSIA VOGELlI, AS AFFECTED BY LATITUDE AND WITHIN-ROW SPACING. Crop Sci. 7: 93-95. (2) BREWBAKER, J. L., and KwACK, 13. H. '. 1963. THE ESSENTIAL ROLE OF CALCIUM ION IN POLLEN GERMINATION AND POLLEN TUBE GROWTH. Amer. Jour•.Bot. 50: 859-865. (3) FRAHM-LELIVELD, J. A. 1957. OBSERVATIONS CYTOLOGlQUES SUR QUELQUES LEGUMINEUSES TROP ICALES ET SURTROPICALES. Rev. de CytoI. et BioI. Veg. 8, pp. 273-287. (4) HUTCHINSON, J. 1926. THE FAMILIES OF FLOWERING PLANTS. 1. DICOTYLEDONS. 328 pp. Macmillan and Co., London. (5) IRVINE, J. E., and FREYRE, R. H. 1959. OCCUURENCE OF ROTENOIDS IN SOME SPECIES OF THE GENUS TEPHROSIA. Jour. Agr. and Food Chern. 7: 106. (6) --- and FREYRE, R. H. 1966. EFFECT OF PLANTING TIME AND PHOTOPERIOD 0;0.; TEPHROSIA VO GELlI. Agron. Jour. 58: 49-51. (7) MARTIN, F. W. 1959. STAINING AND OBSERVING POLLEN TUBES BY MEANS OF FLUO RESCENCE. Stain Techno!. 34: 125-128. (8) POEHLMAN, J. 1\1. .... 1959. BREEDING FIELD CROPS. 427 pp. Holt-Dryden & Co., lnc., New York. (9) ROARK, R. C. 1937. TEPHROSIA AS AN INSECTICIDE--A REVIEW OF THE LITERATURE. U.S. Bur. Ent. and Plant Quar. E-402, 165 pp. ',j 5 t'

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