CHROHOSOKAL AND POLLINATION STUDIES

AS RELATED TO IKTKA-SPECIFIC AND INTER-SPECIFIC

COMPATIBILITY IN THE GENUS PSIDIUM.

A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE

UNIVERSITY OF MWAII IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN HORTICULTURE SEPTEMBER 1967

ByRobert T, Hlrano

Thesis Cossoltteei

Eenry Y. Nakasone, Chairnian Uarujmkl Kaaemoto James C. Gilbert

Ve certify Chet ve have read this thesis and

that in our opinion it la satisfactory in scope and quality as a thesis for the degree of Master of Science in horticulture.

THESIS COMMITTEE

/ Chiinaan

U

TABLE OE CONTiarrSPage

LIST OF TABLES....................................................ill

LIST OP F IGURES................. ivmiRODUCTIOH...................................................... 1UEVIE^^ OF LITERATURE . .......................................... 3

Botanical Aspects ........ . . . * ........................ 3Pollen Germination ........................................ APollination Aspects ........................................ 6Species Compatibility . . . . . . . . . . . ................ 7Chromosome l^umbers ............... 9

>1ATERIALS AND M E T J O D S ........................................... 10

Materials............................................. 10Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . IS

Preparation of Flowers for Pollination .................. 18Pollination............................................ 19Pollen Germination......... 19Chromosome Counts . . . . . ............................ 20

RESULTS......................................................... 21

Pollen Viability .......................................... 21Species Compatibility . . . . . ............................ 27Cltromosome N u m b e r s ........................................ 30Chromosome Morphology ...................................... 37

DISCUSSION....................................................... 40SUMMARY......................................................... 43LITERATURE CITED ................................................. 45

ii

LIST OF TABLESTabic PaceI PERCENT POLLEN GERMIIiATION (LN VITRO) OP PSIDIUM

GUAJAVA L. AT 0, 1 AND 2 HOURS FOLLOWLNG ANTHESIS AND AT 6 LEVELS OF pH USING KNACK’S FORMLTJt............... 22

H PERCENT POLLEN GEIUaKATION (IN VITRO) CP PSIDIDMGUAJAVA L. AT 24 HOURS Il^TERVALS AT 2 pH LEVELS........... 23

III PERCENT POLLEN GERMINATION (IN VITRO) 0? PSIDIUM GUAJAVA L. AT 3 HOUR INTERVALS FROM 27 TO 36 HOURSAFTER ANTHESIS AT 3 pH LEVELS............................ 24

IV EFFECT OF AGE OF POLLEiJ ON FRUIT SET UKEN POLLINATED UPON STIGMAS OF FLOWERS ElASCULATEDAND BAGGED 24 HOURS PRIOR TO ANTHESIS.................... 25

V DURATION OF STIGl'A RECEPTIVITY LN PSIDIUM GUAJAVA L. WHEN POLLINATED AT 24 HOUR ItlTERVALS DURINGA PERIOD OF 4 D.1YS...................................... 26

VI POLLEN TU3E LENGTH AND PERCENT POLLEN GERMINATION OP FIVE PSIDIUM SPECIES USING KHACK'S GSRMI1;ATI0NMEDIA AT pH 5 , 5 .......................................... 28

VII SELF AND CROSS COMPATIBILITY AS DETERMINEDBY FRUIT SET PERCENTAGES................................ 29

VIII PERCENT FRUIT SET OF PSIDIUM GUAJAVA L. CLONESBY HAND POLLIIATION...................................... 31

IX SIZE OF SHORTEST AND LONGEST PAIR OF CUROMOSOMESIN PSIDIUM SPECIES...................................... 38

Hi

LIST OP FIGURESFigure Page

1 I* gpajava. lea ves, flower buds ahd fruits.NOTE SPLITTING OF CALYX (ARROW)...................... 12

2 POLYCARPUM. LEAVES, FL0V7ER BUDS AND FRUITS.EOTE MOSPUOLOGICAL SBIILARITY WITH P. GUAJAVA............ 12

3 P. CUIKEENSE. LEAVES, FLOWER BUDS AND FRUITS.NOTE 3 FLOWERED PEDUNCLE (ARROW) MINUTELYDENTATE CALYX NOT VISIBLE IN PHOTOGRAPH.................. 12

4 P. CUJAVILLUS. LEAVES, FLOWl R BODS AND FRUITS.NOTE 3 FLOVniFj-D PEDUNCLE (ARROW) AND MORPHOLOGICAL SIMILARITY WITH P. GUH^SENSE........................... 12

5 F* CATTLSIANUM VAR. LUCISUM. LEAVES, FLOWER BUDSAND FRUITS. NOTE BARELY VISIBLE OPENING IN CALYXOF FLOWER BUD (ARROW); PETALS BASELY VISIBLE ............ 14

6 F* CAmEIA>R;-M. LEAVES, FLOWER BUDS, AND FRUITS.NOTE MINUTELY DENTATE CALYX AND RAISED PETALS ATAPEX OF FLOI ER BUDS................................... 14

7 FRIEDRICKSTHALIAIRJM. LEAVES, FLOWERS AND FRUIT.NOTE IRREGULAR SPLITTING OF CALYX..................... 14

8 INDONESIAN SEEDLESS PEOGEIJY. NOTE DIVERSE ARRAYOF MORPHOLOGICALLY DIFFERENT TYPES .................... 16

9 CHPJ3MOSOKES OF P. CATTLEIAIRM SHOWING 2n » 7 7 .......... 3310 CHROMOSOMES OF P. CATTLEIANUM VAR. LDCIDUM SHOWING

2n - 66. ARROWS POINT OUT SArTELLITE CHROMOSO>tES . . . . 3311 CfiROMOSOilKS OF P. PRIEDRICBSTHALIANUM SHOWING

2n - 66. ARROWS POINT OUT POSSIBLE SATELLITES.... 3312 CHROMOSOMES OF P. GUINEENSE SHOWING 2n - 4 4 ............ 33

13 CHR01-83S0MES OF P. FRIEDRICHSTHALIANUH SHOTTING 2n - 44 . . 3314 CHROMOSOl-tES OF P. CUJAVILLUS SHOWING 2n - 4 4 .......... 33

15 CHROMOSOMES OF P. POLYCARPUM SHOWING 2n - 22.NOTE SIMILARITY OF CHROMOSOMES WITH THOSE OPP. G P A J A V A........................................... 35

16 CHROMOSOMES OP P. CUAJAVA SHOWING 2a - 22. NOTESIMILARITY OF CiilUJMOSOIffiS WITH THOSE OF P. POLYCARPUM . . 35

iv

Figure Page

17 CKFjOMOSOMES of llsDONSSIAi; SEEDLESS SEEDLINGNUKBER 27 SHOWING 2n - 2 5 ....................... 35

18 CHROMOSOMES OF INDOHSSIAl'I SEEDLESS SEEDLINGNUMBER 15 SHOWING 2n - 2 A ....................... 35

19 CHROMOSOMES OF HORTICULTURAL VARIETY IND0N-ESIAJ3SEEDLESS SHOWING 2n « 3 3 ............................... 35

V

IIJTRODCCTION

The gcnuA Psldlim includes about 150 species of shrubs and trees Chat are native to Tropical Anerlca. Many species produce edible firuits. Of all the edible species known, gualava L. is the ®ost widely exploited for coomercial purposes. Because of its popularity and ease of dissemination, the guava has become a faolllar sight throughout the tropical and sub-tropical world.

Cuava fruits are becoming increasingly important with advances in processing technology (41). However, fruits gathered from the "wilds" and from seedling orchards still make up a large part of the supply for the industry. Differences in the quality of the fruits have made it

difficult to process uniform high quality products. To maintain high

standards in quality it is necessary to develop superior clones for commercial culture (9).

Early clonal selections were primarily from wild guava

populations and not as a result of breeding. Tiie variety J. H.Beaumont (B-30), the commercial variety in Hawaii, was selected from within a seedling population which was established from seeds of a wild selection (8). Today, more emphasis is given to seedling selections from controlled crosses and many horticultural varieties of jP. gualava have been selected (30,40).

The large number of edible species display a vast source of genetic variability; yet, essentially no work has been done on hybridization at the species level. Since genetic variability is

limited within P. gualava. the breeders will eventually have to turn

to Inter-apecific hybrldizatioa for further loprovemanta. To cite an

exanple, India, which relies heavily on the gviava (P. gualava) as a

food source, has been plagued with a very serious guava wilt caused by

Fixsarlum oxysportua f. psldti. Studies by Gauri Shanker (20), Gsuri Shanker et al. (21) and I'vcithani and Srirartava (35) have been conducted to find a good compatible rootstock possessing resistance to the guava wilt. According to these authors, P. cattleianum var. lucldum is known to be free of the guava wilt and is used as one of the rootstocks. Breeding for resistance has not been mentioned. With the vast number of Psldiun species, many of which have never been thoroughly examined, it is likely that a few other compatible species may carry resistant

genes for the guava wilt and other diseases.Although 150 species have been reported, the systematica within

the genus is confused. Taxonomic clarification, particularly from

cytogenetic studies, will provide the breeder with the necessary information to determine breeding procedures.

The study reported herein was undertaken to clarify some of the problems involved in intra- and inter-specific compatibility and also to contribute further knowledge of species relationships through chromosome and pollination studies.

2

REVIEW OF LITERATURE

Although the guava, Psidlum goalava L., has been widely

dlssetalnated throughout the tropical world and have been exploited to a great extent, its closely related edible species have enjoyed little attention In most areas (12). In general, literature on spades other than Psldlma guajava is limited.

Botanical aspects:The genus Psidlum belongs to the family Myrtaceae. It includes

trees and shrubs exclusively from Tropical America. Morphological

descriptions for most of the common species (3,4,12,22,32,36,52) are adequate; however, this is not true for the majority of the other

species, such as polycarpura. gulneense. and culavlllus. The lack of adequate descriptions has contributed to the confusion o f the

systematlcs of this genus.

An example of this confusion la P. gulneanse Swartz which was considered to be synonymous with P_. Araca. by Bailey (3). Bailey lists

gulneenso as the preferred name and K Araca as the synonym, lie also mentions that plants known to the trade as P. gulneense or £. Araca ware usually forms of jP. gualava. L. In 1947 Bailey (4) listed Z.* Araca. Raddi, but makes no mention of gulneense as being synonyaious. however, he Indicated that P. gulneense is only a

horticultural form of P. guaiava. Uphof (52) lists P. Araca as the preferred narao and P.. gulneense. Sw. as Its synonym. He also lists P. fluvlatlle. Rich, with P. gulneensis. Pers. as Its synonym.

These differences In opinion of early systeciatlsts are believed to be the results of classification through Inferences made by

appearance itself. Host of the early taxonomic work did not take Into consideration breeding experiments which could bring to light fundamental genetic assumptions: mainly, that It Is necessary to have stability In environment before Inferences In hereditary differences can be made; that visual observations and comparison do not necessarily show genetic relation of species; and that the basic unit to be

considered should be a genetic or Mendelian population and not restricted to a single type (15).

All of the species studied were consistent with the morphological descriptions given of many authors (3,4,12,32,36,52) with the exception of P. polycarpiin. which seems to fit the brief description of P. polycarptns. LaEib. by Grlsebach (22). Muller (32) describes a species listed as P. polycarpon Al, Anderson. This species Is reported

to originate in the same area as jP. polycarpum Lamb., as described by Grlsebach. Since Muller's brief description seems to fit that of Grlsebach, these two listings could refer to the same species.P. culavlllua could not be found listed In the literature.

Pollen germination:

Pollen gemination studies on P. gualava have been reported by

Balasubrahaanyam, V. R. (5) Desaraty (17), and Nalr, Balasubrahanyam and t3han (34). The media used were generally that of sugar-agar, glucose, and glucose plus sugar mixture ranging from two to twenty five percent sugar. They found that the germination percentage depended

4

upon the variety tested and a range fron 40.3Z to 93.8% was obtained. Optirauas at different sugar levels were recorded for different varieties. Sehgal (42), however, found 15% sugar solution best for optiaum germination in three cultivars of P. gualava. Ue also found that pollen tube length increased up to 25% sugar solutions.

I^ack (29) concluded that optimal pH range for pollen growth varied with species. Zielenski (54) reports that magnesium was found to be effective in promoting rapid germination and tube extension. He

also found that it eliminated the bursting of pollen tube. Martin and Ortiz (33) reports that sweet potato pollen failed to germinate in vitro under any circumstance. They also make reference to ’‘pseudogcrmlnatlon'* a term used to describe the evacxiation of cytoplasm from pollen grains without the t\ibe wall in a manner resembling germination.

Although pollen germination in ?. guajava has been studied for

a long time (5,34,42), it is only recently that the role played by the various ions has become recognized. Brink (11) showed that Ca*** alone stimulated pollen germination. In germination studies which Included pollen of cualava Kwack (29) and Brewbaker and kwack (10) also found Ca'*' ion in addition to Boron to be a major factor in

promoting pollen germination and tube growth. Calcium ions enhanced growth by its presence, especially when Mg, K or Na ions are present

(10). Optional concentrations for these ions in germination arc given by Kwack (29) as:

5

Sucrose lOZ

Boric Acid 100 ppmCa(NOg)2:4R20 300 ppmMg S04:7H20 200 ppmK NO3 100 ppm

Ha found these concentrations to be optional for many species, iie also found that concentrations above and below these levels decreased growth. Kwack (23) found that when levels of Hg or K ion varied from their optimum, pollen growth decreased.

Pollination aspects;Although Dasarathy (17) believed that cross pollination in the

guava is the rule in nature, the mode of reproduction of iuialava L. is described by Dodds (18) as being partial cross-fertilization.Studies condiu:tcd fay Soublhe and Gurgel (49) show that the average extent of cross-pollination was about 35.6%, thus confirming Dodds’ hypothesis. Seth (44), however, found sorae cultivara of Rualava to be totally cross-incompatible. Uc also found others to possess

degrees of cross-incompatibility and that although the pollen grains germinated on the stigma, tube growth was inhibited in the style.

Seth (43) also found this to be true in the cultivar Seedless. This suggests a gamctophytic type of incompatibility.

Early studies by Balasubrahmanyan (5) concluded that the stigma

became receptive 2-3 hours after floral anthesis and remained functional for 48 hours, while the pollan grains remained viable for only 4 hours after dehiscence. Sehgal (42) reported tiuit the pcllen

was found to bo viablo for 24 hours.

Species compatibility;

Inter-specific cross-incompatibility has been recorded only among three Pstdlum species; gualava L., jP. cattleianum Sab. and P,. molleBertol (14,44). Extensive work has not been done on inter-specific hybridization because sterility is usually involved. In general it is quite common for inter-specific hybrids to be sterile either by genic or chromosome disharmony. Failure of hybrids is generally caused by hybrid inviability or hybrid sterility.

Triploids could be created by controlled hybridization but, unfortunately, none have bean reported. They nonaally tend to be sterile or partially sterile. Singh (46) concluded that seedless guava

was of no importance commercially primarily because of poor bearing.

It is generally agreed (6,7,13,24,26,45,56) that 4n x 2n crosses usually give better seed set than the reciprocal 2n x 4n crosses which are usually incompatible,

Wagner and Ellison (33) working with crosses between diploid and autotetraploid Aspara'jus officinalis L. and Beasley (6) with American 26-chrono80me and Asiatic 13 chromosome species of Gossypium found exceptions when only the 2n x An crosses produced seeds.

The occurrence of tctraploid seedlings of Paldium ffua1ava is not too uncommon. Nel (37) reported a tetraploid seedling found in his breeding program. Gauri Shanker et al. (21) reported selection of a tetraploid P. gualava from among a diploid seedling population.

Literature on the breeding of these tetraploids to produce triploids

have not appeared.

Trlplolds of ?. gualava have been recorded by Kumar and Ranade (27)

and Hajuiader and Singh (31). Apparently, many more triploida exist today and the cultivar Indonesian Seedless is one of them.

Seth (43), reported that the cultivar Seedless was a diploid ofhybrid origin for it possessed abnormal melosis and irregular pollen grains with low viable percentage. Kajunder and Singh (31) agree with ICumar and Ranade (27) that seedlessness in guava is due toautotriploicly and they believe that Seth’s count of 2n - 23 was the

result of misinterpreting the trlvalant configurations as bivalents.If this is the case, then the diploid hybrid mentioned by Seth (43) was actually a triploid.

In general, most workers (21,23,37,39) feel that tetraploldy is of great importance in the breeding program of guava (P. gualava). Ihe

results of 4n x 2n crosses would give a vide selection of trlplolds

which would be seedless or possessing only a few seeds. Also, if the

tetraploids have some Important characters, their value as parents would be enhanced. Superior triploid selections would be of great importance for P. ;;tua1ava can be easily propagated vegetatlvely.

Singh (46) on the other hand, believes that seedless guava varieties would be of no commercial importance because of unfavorable characters such as irregular fruit shape, poor bearing, inferior flavor and taste, he agrees that seedless cultlvars may prove useful in breeding.

hybrid inviability is reported by Allard (1) to be caused by

single gene lethals, by general incompatibility of parent genotypes.

8

and by unbalance of embryo and endosperm.Thompson (51) believes that the primary reason for using the

larger chromosome numbered plants as the female parent, is to obtain a greater percentage of the chromosome complement of that parent In the

endosperm.

Chromosome numbers:Chromosome numbers have been reported for: Psidlum gua1ava L.

2n - 22 by Atchison (2), D'Cruz and Rao (16). Also, a seedless variety of gualava has been reported by Kamar and Ranade (27) in India as

having 33 chromosomes. D'Cniz and Rao (16) also reported counts of 21, 22, 30 and 33 for aneuploids of Psidlum gualava. Tetraploid of P. gualava has also been reported by Nol (37) Gauri Shanker et al. (21). Chromosome counts of P.. cattlelanum and P.. cattlelanlum var. lucldum

have also been reported to be 2n - ca. 88 (2,47).Chromosome numbers for the other species have not been found

in available literature.

9

MATERIALS AND METHODS

MATERIALS

Guava plant materials were obtained from the Hld-Pac Experimental Farm at the University of Hawaii and also from the Poamobo Experimental Farm. The Paldium species used In this study along with their descriptions are as follows;

Psidlum Rualava Linn. (Fig. 1). The common guava— introduction date unknown; well-established in the wild state. Leaves to

6 inches long, pubescent beneath, veins costatc and prominate beneath; peduncles 1 to 3 flowered, usually 1; calyx closed in the bud and split prior to anthesis into

4 to 5 persistent lobes, usually 5; fruits to 4 inches long, yellow when ripe with white, yellow or pink flesh and shapes vary from a globose-ovoid to pyriform.

P. polycarpuTO Lamb. (Pig. 2). Introduced in March 1965,Accession number 7519, strongly resembling P. Ruajava L. Leaves to 6-7 inches long, pubescent beneath, veins to costate and prominent beneath; peduncles 1 to 3 flowered but usually 3 flowered; calyx closed in the bud and splits prior

to anthesis into 3-5 persistant lobes, usxially 4; fruits to 4 inches long, pyriform, yellow when ripe with pink flesh.

11

PLATE I*

Figure 1 Paldlna guajava. Leaves, flower buds and fruits. Notesplitting of calyx, normally Irregular.

Figure 2 Paldlun polycarpum. Leaves, flower buds and fruits. Notemorphological elmilarities with P. gualava.

Figure 3 Psldiua gulneenae. Leaves, flower buds aixd fruits. Note 3 flowered peduncle (arrow).

Figure 4 Psldiua culavlllus. Leaves, flower buds and fruits. Notemorphological siiailarlties with P. gulneensa. 3 flowered peduncle obvious (arrow).

*1/3 normal size

13

PLATE II*

Figure 5 Pslditm cattlelauum. Lsaves, flower buds and fruits. Notedentated calyx and petals slightly raised at apex of flower bud.

Figure 6 Psidlum eattleianum var. lueiduni. Leaves, flower buds andfruits. Note minutely dentated calyx and petals not raised above opening of bud.

Figure 7 Psidtum Friedrlehsthalianura. Leaves, flowers and fruit.Note Irregular splitting of calyx.

*1/3 normal size

14

-IK

i-

15

PLATE III*

Figure S Progeny of cultlvar Indonesian Seedless. Shown here are 9 distinct morphological types.

*Figures reduced 1/6 normal size

Z.* gulneense Swartz. (Pig. 3). The Brasilian guava, introduced

November 1964, Accession number 7463, strongly resembling

£• guineense. Leaves to 5 inches long, pubescent below, with

prominent veins; peduncles 1 to 3 flowered, usually 2 or 3; calyx minutely dentated and splits into 3-5 lobes at anthesis; fruits to 1 1/2 inches long, globose-ovoid, greenish yellow when ripe with white flesh.

cu1avlllus (Fig. 4), Introduced in early 1960’s. Leaves to 5 inches long, pubescent below with prominent veins; peduncles 1 to 3 flowered, usually 3; calyx tdnutely dentated, xisually

splitting into 5 lobes at anthesis; fruits to 1 inch long, globose-ovoid, yellow when ripe with white flesh.

£• cattleianum (Fig, 6). The strawberry guava— introduction date

unknown; found wild in parts of Hawaii. Leaves to 3 Inches long, glabrous; peduncles short, one flowered. Calyx

dentated, near apex, petals exposed and slightly raised above calyx, calyx splits at anthesis into 4 to 5 lobes, usually 4; fruits to 1 1/2 inches long, globose-ovoid; red to purplish red when ripe with white flesh.

cattleianum var. lucldum (Fig. 5). The yellow strawberry guava— introduction date unknown; found wild in parts of Hawaii. Leaves to 3 inches long, glablous; peduncles short, one flowered; calyx minutely dentated, opening at apex, exposing petals, sometimes nearly round, calyx splits at anthesis into 4-3, usually 4 persistent lobes; fruits to 1 1/2 inches long, globose-ovoid, yellow when ripe with

17

white flesh.P. Frledrlchsthalianum Sledenau (Fig. 7). Introduced from El

Salvador in 1956; introduced from Costa Rica in Septecber 1964. Leaves to 3 inches long, glossy above, pubescent below; peduncles one flowered; calyx closes in bud. Irregular splitting, usually into 2 lobes at anthesis; fruits to 2 1/2 inches long, yellow to greenish yellow when ripe with white flesh.

Horticultural varieties of P. gualava \ised in this study include

the Florida selections Patillo and Pink Acid and University of Hawaii varieties Kong Kong Pink and J. U. Beaumont.

A progeny of 36 open pollinated seedlings from Indonesiiin Seedless were grouped into 12 distinct morphological types. A representative

plant from each of 9 groups is shown in Figure 8. Plants of some groups possess vegetative characters distinctly different from

P. gualava.

METHODS

Preparation of flowers for polllnatloa;Flowers were emasculated the day prior to anthesis. This was

done carefully by removing portions of calyx, petals and anthers at the base of the disc with a sharp knife without damaging the style.The flowers were then bagged with glycine bags and labeled with

appropriate tags. Flowers to be used as pollen source were also tagged and bagged one day prior to anthesis.

13

Pollination;In testing for coiapatlblllty, the emasculated flowers were tagged

and pollinated with the desired pollen on the "normal" day of anthesis using forceps to place the pollen on the stlgaa. Compatibility was determined by fruit set 30 days after pollination.

Polllimtlon to test for stigma receptivity was done at 24-hour intervals beginning at 24 hours and extending up to 96 hours after emasculation. Fresh pollen were used for dally pollinations.

In testing for pollen viability (in vivo) pollination was dona by

using pollen collected at floral anthesis and kept in a vial at room

temperature. For the first 24 hours pollination was conducted at 6-hour intervals. This was followed by pollinations at 3-hour

intervals, up to 48 hours after collection. Last pollination was done when tha pollen was 72 hours old. Flowers \;sed for this study were emasculated 24 to 48 hours prior to pollination.

Pollen germination;Pollen viability studies (in vitro) wore conducted by using a

medium prescribed by Kwack (29). It consists of 101 sucrose, 100 ppm Boric acid, 300 ppm Ca(N03)2.4 H2O, 200 ppm MSSO4.7K2O, and 100 ppm KKO3. The medium was adjusted to pU 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0 with O.IN NaOU and/or O.lli! H2SO4.

For studying the duration of viability, pollen of J. E. Beaumont,

a cultivar of F. Rualava was used. Pollen were sown on a drop of laedlum on a glass slide and allowed to germinate in a saturated atmosphere for a period of 6 hours. Sowing was done by dusting the

19

pollen grains from tha anthers with a camel’s hair brush onto the

medium. Flowers used for pollen source were bagged 24 hours prior to anthesls to avoid contamination. Different levels of pH were used and

pollen grains were sown at different time Intervals. Three replicates were used.

Chromosome counts;

Available guava species were transplanted into gallon cans using

vermicullte medium. The plants were sprayed weekly with foliar fertilizer. Cuttings were also used to obtain roots. These cuttings,

usually green-wood, were treated with 2,000 ppm SNA/talc mixture and stuck in vermicullte in tha talst box.

Hoot tips were used for chromosome counts and karyotype studies. Root tips were collected between 10 a.m. to 12 a.m. for it was found that a high percent of dividing cells were present during this period.

They were immediately Immersed in 0.002 M 8-hydroxyqulnoline for four to five hours and kept at 45 to 50“ F. They were then fixed in Camoy'a solution (1:1:2). After fixing, they were hydrolized with a 1:1 12K KCl-alcohol mixture from 2-3 1/2 minutes. The root tips were then stained with a IZ aceto-orccln solution and squashed, following the methods described by Kamemoto ct al. (25).

Statistical treatment;Confidence interval tables, Poisson distribution and standard

error methods as outlined by Snedecor (48) were used for analysis of data.

20

RESULTSPollen viability;

Pollen grains of Beaumont (P. .guajava) sown soon after floral anthesis germinated well at all pH levels except pH 7. It should ba mentioned here that anther dehiscence occurs about 30 minutes prior to floral anthesls. This discrepancy was not noted In pollen grains sown an hour later when all pH levels gave excellent germination (Table I). As the age of the pollen increased, its germination percentage

generally decreased from 74.0% and 76.1% at pH 4.5 and 7.0 respectively, to zero at 48 hours of age (Table II). It appears from

the data in Table III that higher pH enhance germination of older pollen grains. It seema likely that pollen 27 hours and over in age do not lose viability entirely or substantially but that viability Is affected by changes in pH. In vivo pollination studies confirmed the findiiigs that pollen of guajava remain viable after 48 hours follow­

ing floral anthesls (Table IV), At 48 hours, three out of five pollinations set fruit and at 72 hours, one fruit set out of five was recorded. This finding is contrary to those of Balasubrahmanyas (5) and Sehgal (42) who found pollen to be viable only for 4 hours and 24 hours, respectively.

Stigma receptivity studies in the field confirmed the findings of Balasubrahmanyam (5) and Sehgal (42). Table V shows that at 48 hours following floral anthesls, nine out of 10 flowers pollinated set fruit, and at 72 hours, 10 attempts did not set a single fruit. Maximum duration of stigma receptivity Is believed to be somewhere between 48 and 72 hours following floral anthesls.

21

22

TABLE I. PERCENT POLLEN GERKINATION (IN VITRO) OF PSIDIUM GUAJAVA L. AT 0, 1 AND 2 HOURS FOLLOWING ANTHESIS,

AND AT 6 LEVELS OF pH USING KWACK*S FORMULA

pliHours after Anthesis

Percent Germination Observed

4.5 0 1001 1002 99.8

5.0 0 99.61 1002 93.7

5.5 0 99.31 98.82 97.2

6.0 0 99.31 1002 99.7

6.5 0 97.51 97.22 99.8

7.0 0 36.31 91.02 85.9

23

TiVELE II. PERCENT POLLEN GERMINATION (IK VITRO) OF PSIDIUM GUAJAVA L. AX 24 HOUR INTERVALS AT 2 pH LEVELS

pHHours after Arthasls

Percent Germination Observed

Confidence Interval 95 Percent

4.5 0 100.024 74.0 68.28 - 79.83

48 0.07.0 0 36.3 30.30 - 41.70

24 76.1 68.94 - 82.06

48 0.0

24

TABLE III. PERC3OT POLLEN GEP.MINATIOIl (IN VITRO) OP PSIDIUM GUAJAVA L. AT 3 EOUR INTERVALS FROM 27 TO 36 HOURS AFTER AI3THESIS AT 3 pH LEVELS

Hours after Percent Germination Confidence IntervalpH________ Anthesls____________ Observed_______________ 95 Percent_____5.0

6.0

7.0

27 030 1.1 0.20 - 3.0733 0.3 0.03 - 1.1136 0.5 0.05 - 1.80

27 030 0.8 0.08 - 3.0233 1.6 0.43 - 3.5736 0.3 0.02 - 1.6227 75.4 68.36 - 80.3630 51.8 46.09 - 57.10

33 74.4 67.96 - 80.7236 IS.6 14.12 - 23.20

25

TABLE IV. EFFECT OF AGE OF POLLEN ON FRUIT SET WHEN POLLINATED UPON STIGMAS OF FLOATERS aUSCULATED AND BAGGED

24 HOURS PRIOR TO ANTHESIS

Age of Pollen Hours after Anthesis

Number of Flowers Pollinated

Number of Fruit Sat

0 (at anthesis) 5 56 5 512 5 518 5 424 5 530 5 536 5 542 5 548 5 372 5 1

26

TABLE V. DLTJATION OF STIGMA RECEPTIVITY IN PSIDIUM GUAJAVA L. WHEN POLLINATED AT 24 HOUR INTERVALS DURING A PERIOD" OF 4 DAYS

Hours after Anthesis*

Kuober of Flowers Pollinated

Kuniber of Fruit Set

0 10 1024 10 1043 10 972 10 0

*Flovers were emasculated 24 hours before anthesis.

Data presented in Table VI shows the results of pollen germination studies which verify the fact that all the species utilized in the crosses possessed viable pollen with the exception of P. cattleianum var. lucldum. Pollen from var. lucldum did not geminate on the medium utilized or even in two to six percent sugar solutions. Although pollen germination was not observed, the pollen appeared to be viable as they were plutap and well shaped. Also, crosses utilizing pollen of

cattleianum var. lucldum gave good fruit set percentages and seed

set. Indicating normal viable pollen.

Species compatibility;Table VII shows that reciprocal crosses were obtained from crosses

between P. gualava x P. cu1avlllus and between cattleianum xP.. cattleianum var. luciduta. gualava x P,. gutneenae cross was successful but the reciprocal was not. The cross between P,. gulneensa X £. cu1avlllus was successful, but, unfortunately, the reciprocal was not made. Results of other species crosses and their reciprocals are shown in Table VII.

Inter-specific crosses of the other species Involved were not successful. The number of crosses made depended upon the number of

flowers available on a given day. In soma cases it was difficult tomake as many crosses as would have been desirable.

The species P,. Friedfrichsthaliaaum used in the crosses was from El Salvador with chromosome nuaiber of 2n « AA, and not the Costa Rican P. Friedrichsthaliaaum with 2n 66.

27

28

TABLE VI. POLLEN TUBE LK^OTK MID PERCENT POLLEN GER.MINATION OF FIVE PSIDIUM SPECIES USING

^JACK’S GEB^aKATION MEDIA AT pH 5.5

Percent Germination Observed

TubeLengthMicrons

P. ciilavlUus 98.0 ‘ 0.80 440 * 62

£• Friedrichs thallanim 37.1 * 1.73 310 * 24p. gulneensa 49.2 * 5.25 420 ‘ 36p. CEttlelanjiin 32.0 * 2.74 260 * 38

p. catclelaaum var. lucldum 0 -

p. s?,ua1ava var. Hon?. Kona Pink 72.2 ‘ 1.39 350 * 38p. Kuaiava var.

Indonesian Seedless 2.4 » 0.37 420 * 52p. gualava var. B-30 99.3 * 0.46 500 * 24

TABLE VII. SELF Al® CROSS COI^PATIBILITY AS DETERMINED BY FRUIT SET PERCENTAGES®

29

Female Parent 1 2Male3

Parent4 5 6

1. P. Rualava 36 0 0 0 1 02. P. cattleianum 0 38 33 0 0 03. P. cattlelanxia var.

lucldum0 56 79 0 0 -

4. P. Kulneenae 25 0 0 6 100 05. P. culavillus 50 0 0 0 40 06. P. Friedrichsthallanum 0 0 0 0 0 2®

®Actual number of flowers pollinated ranged from 5 to 221.^Species acro&s the column are represented by numbers corresponding to the numbers before each species in the first column.

^Obtained by sib mating.

Intra-speciflc crosses gave favorable results although ooly six

cultivars were utilized In the study (Table VXII). When Indonesian Seedless was crossed with Hong Kong Pink, no fruit set was obtained.The reciprocal cross was also unsuccessful. The lack of fruit set from the cross, Uong Kong Pink x Indonesian Seedless, was expected; for noraally, trlplold pollen is either Inviablc or have a very low I>ercentage of viable seeds as shown in Table VI. Although no fruit set was recorded when Indonesian Seedless was used as the female parent. It should be noted that open pollinated fruits containing a

few seeds have been obtained. These fruits could be Che result of self or sib DuiClng. However, it is very unlikely for preliminary

studies showed that when used as the pollen source In controlled crosses, no fruit set was obtained. In a few Instances when Indonesian Seedless was used as the female parent, fruit set with a few viable seeds were produced.

All the other crosses involving plants with 22 chrosK>somes

performed as expected with fruit sets ranging from 80 to 96.71.

Chromiosomes numbers tP. gualnva was found to be 2n - 22. This confirms the somatic

number reported earlier by other workers (2,16,27). Indonesian Seedless was found to have 2n ■ 33 (Fig. 19). Chromosome counts were made for three seedlings of Indonesian Seedless (2n ■> 33) x jP. gualava

(2n •* 22) cross. Somatic numbers of 2n “ 21, 24, 25 were recorded, clearly Indicating an aneuplold series resulting from a trlplold x

diploid cross. Figures 17 and 18 show chromosomes of Indonesian

30

31

TABLE VIII. PESCEllT FRUIT SET OF PSIDIUM GUAJAVA L. CLONES BY KAKD POLLINATION

Nursber of Flowers Crossed

Nttsber of Fruit Set

Percent Fruit Set

Indonesian Seedless X Hong Kong Pink 30 0 0.0

Hong Kong Pink X Indonesian Seedless 30 0 0.0

Patillo XHong Kong Pink 30 29 96.7

Hong Kong Pink X Patillo 30 28 93.3

U of n Selection 029 X Allahabad Safeda 15 12 80.0

Allahabad Safeda X U of H Selection 029 15 13 87.7

32

PLATE IV*

Figure 9 Chronsosomcs of P. cattlelantm with 2n ■ 77.Figure 10 ChromosoECS of cattleianum var. luetdua with 2n ■ 66.

Note chromosome pair with satellite (arrow).Figure 11 Chrooosomes of P. Friedrichsthalianuia with 2a - 66.

Note fragment or satellites (arrows).Figure 12 Chromosomes of P. gulneense with 2n - 44.

Figure 13 Chromosomes of P,. Friedrichsthalianma with 2n ■ 44.

Figure 14 Chronosomas of P,. cui a villus with 2n - 44.

*A11 figures magnified 7740 X

33

J

10

f

T = ^ V ' ' ‘

i

-\V r'S ir t

c » s • . > *

r . -

■ 1 2V

r

J v"

.• ff/

13

»*>iS *!

I

11 1 4

34

PLATE V*Figure 15 Chromosomes of P, polycarpua with 2n - 22. Note

slnilarities of clirooosoaes with those of P. eualava.Figure 16 Chrotsosotaes of ttualava with 2n 22. Note

similarity of chrotaosoEes with P. polycarpura.Figure 17 Chronosoaas of Indonesian Seedless seedling nurdjer 27

with 2n ■ 25.Figure IS Chromosomes of Indonesian Seedless seedling number 15

with 2n - 24.

Figure 19 Chroioosomes of variety Indonesian Seedless with 2n “ 33.

*A11 figures magnified 7740 X

35

15

" a

- c

! / r I

If

%

17

18

/

V:-"

V v - \

M -

16 19

Seedless seedlings, numbers 27 and 15 respectively. D'Cruz and Rao (16) also reported 2n nuiabers of 21 and 30 for aneuplolds of gualava.

The species Introduced as P. polycamua (Fig, 2) is believed by

the author to be gua-java (Fig. 1) and it too was found to possess 2n ■> 22. Besides having the same chrocosoms number, these two species are indistinguishable.

Chromosome nunibors of P. cattleianua and its botanical variety lueidum were found to differ from the 2n ■ 88 reported by Atchison (2), and Smith-White (41). Two plants of P. cattlelanum investigated showed chromosome numbers of 2n - 77 (Fig. 9). P. cattleianum var. lucldua. on Che other hand, was found to be 2n « 66 (Fig. 10). This number was determined for one plant only. Also observed for P.. cattleianum var, lucldum were two chromosomes with oatellitea (Fig. 10). Atchison (2) also obseirved a pair of satellite chromosomes.

P. "uiceense and a species introduced as P. culavlllus were found to possess a somatic complement of 2n 44. These two species appear

Co possess cany similarities in vegetative characters. Figure 12 shows the chromosome complement of P. Rutneense. while P.. culavtllua chromosomes are shown in Figure 14.

Figure 13 shows that the species Introduced from El Salvador as

P* Friedrichsthalisnura possessed 44 cliromosoces, However, two plants from a batch of seeds introduced as P. Friedrichsthalianum from Costa Rica were found to possess a somatic number of 2n > 66 (Fig. 11). What

appears to be 2 chromosome fragments or saCelllCes were also observed (Fig. 11). These plants strongly resemble the plants from El

Salvador. Unfortunately, flowers and fruits from the Costa Rica

36

introductions vcre not available for detailed comparison.

Chromosome norphology;

Majority of the chromosomes of £. eattlelrnia appeared to have median centromeres as shown in Figure 9. Its shortest pair of

chromosomes averaged 1.30 microns, while the longest averaged 2.69 microns.

Examination of chromosomes of cattleisnum var. lucidum showed the presence of a pair of satellite chromosomes (Fig. 10). The other pairs appeared normal, mostly with median contromercs. The mean

lengths of the shortest and longest pair of chromosomes were 1.34 and 2.42 microns, respectively. Tabic IX chov/o the mean lengths of the shortest and longest chromosomes of all the species studied.

Chrottosoma figures shown in Figure 11 for the Costa Rican P. Frledrichsthallantnn show what appears to be chromosome fragments or

a pair of satellite chromosomes. It seems more likely that it is a

pair of satellite chromosomes as one of the fragments seems to be attached to a chromosotr«. The average lengths of its longest and

shortest chromosome pairs were found to be 1.34 and 2.75 microns.Satellites were not observed in P. Friodrlchsthaliaaura from El

Salvador (Fig. 13). Chromosome lengths ranged from 1.20 to 3.26

microns for its shortest and longest chromosome pairs, respectively. Chromosomes of P.. gulneense are shown in Figure 12. Its shortestL

and longest pairs of chromosomes were found to bo 1.65 and 3.66 microns, respectively.

37

38

TABLE IX. SIZE OP SHORTEST AMD LONGEST PAIR OP CHROMOSOMES K PSIDIUM SPECIES

Shortest(Microns)

Longest(Microns)

P. cattlelanvm 1.30 ‘ 0.04 2.69 * 0.53p. cattleianutt var. Incldun 1.34 * 0.08 2.42 * 0.14

£• Friedrichsthalianum (2n • 44) 1.20 * 0.02 3.26 * 0.03p. Priedrichsthalianua (2n - 66) 1.34 * 0.05 2.75 * 0.21p. gulncense 1.65 * 0.01 3.66 * 0.16p. cu1avlllus 1.46 * 0.06 2.70 * 0.08p. polvcarpxia 1.91 * 0.08 3.31 * 0.17

p. gualava 1.83 * 0,18 3.64 * 0.09

£• culavlllus appeared to have more chromoaomes with ncdian

centromeres than P. gulneense (Fig. 14). Its shortest and longest chrorosome pairs were 1.46 and 2.70 microns, respectively.

Mean lengths of 1.91 and 3.31 sdcrons were measured for the shortest and longest chromosome pairs of P. polvcarwua. Tliere appears to be morphological similarities between its chromosomes and those of P. walava (Fig. 15).

£.• guclava was found to have mean chromosome lengths of 1.83 and 3.64 microns for Its shortest and longest pairs. Its chromosome complement appears to be identical to that of polycarpum.

39

Tlie results of this study have shown that there is need for further clarification in the systeisatics in the genus Psidlua. especially through a cytogenetic approach.

The finding of 2n ■ 77 in 2 plants of the species classified as P. cattleiantna has given rise to a feu questions. For Instance,P. cattleianinn has previously been reported to have a sonatlc complensent of 2n "> ca. 83 by Atchison (2) and Saith-T^lte (47). This number would seem more logical if, as it is believed, the basic number for the genus is >I " 11. It is also a well-known fact that polyploids with even chromosome numbers perpetuate themselves with greater ease than do

polyploids with odd numbers. The two plants studied were found to have

a chromosome complement of 2n ■ 77, making then heptaploids. This seems unusual since heptaploids do i;ot normally perpetuate themselves because of a high degree of sterility. Unfortunately, the plants

used for pollination studies were not the same plants used for chromosome counts. However, these plants all came from a self­pollinated progeny of P.. cattleianua.

A probable explanation of good fruit set and viable seed production of the plant known hers as P_. cattleiaaum is the hybrid origin of this species which acquired the ability to perpetuate Itself parthenocarpically, thus producing apomictic seedlings

retaining tha parental complement of 2n ■ 77. Evidence to support this hypothesis can be derived from reciprocal crosses between P,. cattlelanum and its botanical form, lucidum. If seedlings from

DISCUSSION

these crosses ere apomictlc in origin, uniform progenies resembling the female parent should be obtained. Such crosses have been made for further studies to substantiate the hypothesis presented above.

As previously mentioned, the trees of P. cattleianum used for compatibility studies were not positively known to be of the same origin as those from which chromosone counts were made. Self pollination in this study gave a 382 fruit set. If all the plants came from the same origin, then this would indicate a high degree of parthenocarpy.On the other hand, if the plants came from a different source, then it would indicate that the plants used for chromosoiBe studies could possibly be hybrids.

Z* cattleianum var. lucidum has been reported as 2n ■ ca. 88.This study revealed that the plant known here as cattleianum var.lucidiTOi possessed a oomatlc complement of 2n ■ 66. Unfortunately, only one plant was used in determining the chromosome number of this species. Because of the discrepancies in chromosome ntnnbers, examination of a larger population of var. lucidum has become necessary.

Chromosome studies of P.. Friedrichgtholtemum suggest aither that two or more races are present within the species, or that the Costa Rican plants and those from El Salvador are different species. The

plants from El Salvador were found to have 2n « 44, while those from

Costa Rica possessed a 2n - 66 complement. Morphologically, these

plants are very similar and difficult to differentiate. The Costa Rican plants still have not flowered to be compared more closely.

Z* guineenae was found to possess 2n - 44. The same number was also found in culavillus. Since these two species appear to be

41

\A2

very similar morphologically and the fact that they cross readily, the possibility exists that P. culavlllus is just a variety or form of Z* wlncense.

Tlie somatic number of 2n - 22 was found in both P_. polycarpum and jP. gualava. P. polycarpvm is thus believed to be a variety of Z* g.ua1ava. not only by chromosome number alone, but also by similarity in chromosome morphology and vegetative characters. Its resemblance to P. gualava la so great that one would not be able to differentiate it from P. gualava.

The cultlvar Indonesian Seedless (P. gualava) was found to have a very low percentage of viable pollen. Cytological studies showed it

to be a trlploid. This explains its compatibility habits. The cross,P. gualava'Hong Kong Pink' x P. gualava 'Indonesian Seedless* failed

to set any fruit, but the reciprocal occasionally sett fruits with viable seeds. Seedlings of Indonesian Seedless have shown a wide range of differences in morphological characters (Fig. 8). Chromosome numbers of two of these seedlings showed 2n • 24 (Fig. 18) and 25 (Fig. 17). These numbers correspond to those reported by D'Crur and Rao (16).These numbers indicate the probable existence of a series of aneuploids for P. gualava in the natural state.

SUtaiARY

1. Studies on pollen viability, stigraa receptivity, species

compatibility, and cliroioosoaie counts are presented for Pstdiua species.

2. Pollen germination and viability were found to extend up to 72 hours after floral anthesis in vivo but limited to 48 hours in vitro. No apparent species differences in pollen germination could be noted except that of P. cattleianum var. lucldum which could not be germinated.

3. Chromosome counts were made of cattleianum (2n «■ 77),£• cattleianum var. lucldum (2n - 66), P. cujavlllus (2n « 44),P. gulneense (2n •• 44), P. Frledrichsthallanum (2n • 44,66),P.. polycamumt (2n 22) and several cultivars of P.. auaiava(2n - 21,22,24,25,33).

4. The following inter-specific hybrids were obtained: JP. gualava x

euiavlllus. £. cuiavlllus x jP. gualava. P. gulneense x P. guajava. P. guinaense x P. eu1avlllus. P. cattlftianum x £. cattleianum var. lucldum. and P.. cattleianum var. lucldum x P. cattleianvm.

5. Inter-specific incompatibility was noted between; gualava x H* cattleianum and its botanical variety lucldum; J P . gua1ava x P. Friedrichsthallanutt; J P . cattleianum and its botanical variety lucldum X P.. gulreense. P. cui avlllus and P. Priedrlchsthalianum; J P . gulneenae x gualava. 7_. Frledrichsthalisnum; P. cu1 avlllus

X JP. Frledrichathalianum; Frledrichsthallanum was found to be

incompatible with all the species involved in this study.6. cuiavlllus and gulneenae seem to be the same species, and

P. polycarpum seems to be the same species as P. gualava.

44

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AS