Annals of the Sri Lanka Department of Agriculture. 2008.10:117-128.
STUDIES ON DEVELOPMENT OF HERMAPHRODITE INBRED LINES AND THEIR MAINTENANCE IN F1 HYBRID PRODUCTION OF PAPAYA (Carica Papaya L.) K.D.A. PERERA, R.H.W. DISSANAYAKE and H.D. JAYAWICKRAMA Fruit Crops Research and Development Centre, Kananwila, Horana
ABSTRACT An investigation was conducted to develop gynodioecious inbred lines of papaya and to study their maintenance as inbreds in F 1 hybrid production. A total of 98 accessions were studied to develop inbred lines by selecting hermaphrodite trees and selfing for generation advancement. Two lines CP1-2 and CP 13-29 reached uniformity in most of the desirable growth characteristics in 3 rd generation (G3). The χ2 values computed showed that these two lines reached the sex ratio of 2 hermaphrodites to 1 female in the 4th generation. Also the fruit flesh colour which was segregating in CP1-2 reached uniformity in G4. Stability of the characters was confirmed in G 5 suggesting that these lines could be utilised as inbreds in F1 hybrid production. Both lines produced predominantly elongated type of hermaphrodite flowers but were found to have few petandria type hermaphrodite flowers and male flowers during certain months of the season. In order to maintain these lines, another experiment was conducted to study the success of fruit setting of these two types of hermaphrodite flowers under flower bagging and without bagging. Elongated type flowers had around 72% fruit set under bagging and without bagging while in petandria type, the success under bagging was significantly lower (2%) to that of without bagging (40%). This showed that bagging flowers alone would not be sufficient but identifying the correct flowers and hand - pollination are required to have successful fruit setting in maintaining the inbred lines. However, a detail investigation is required to study sex reversal during the season. KEYWORDS: Flower types, Fruit flesh colour, Self pollination, Sex reversal.
INTRODUCTION Cultivation of papaya in a commercial scale has increased in Sri Lanka over the past few years with the release of ‘Rathna’ variety by the Department of Agriculture and introduction of ‘Red lady’ by the private sector. The variety Red lady is a F 1 hybrid in which the seeds are imported and distributed among farmers where as the Rathna is an open pollinated variety and seeds are produced by selfing. Both varieties are highly productive and popular among growers in spite of the few drawbacks inherited in these varieties. The development of our own varieties is important to fulfil the growing demand for seeds and to avoid the risk involved in the dependency of the introduced varieties. In such a situation, effort should be made in developing F1 hybrids too as they are expected to be highly productive due to heterosis. Heterosis in papaya has been exploited in other countries and superior hybrids have been developed with better yield and quality (Subramanyam and Iyer 1984; Chan 1992; Singh and Sharma 1996; Ram, 1982; Dinesh and Yadav, 1998; Ram et al., 1999). In Sri Lanka, unavailability of true - to - type varieties is one of the major constraints to initiate a hybrid breeding programme in papaya. There are three basic sex types in papaya i.e. staminate, pistillate and hermaphrodite. Development of hermaphrodite lines is advantageous as every tree in the progeny is a bearing plant with hermaphrodites and females in the ratio of 2:1 (Storey, 1976). Hence the
118 PERERA et al.
present investigation was undertaken to develop hermaphrodite inbred lines in order to produce F1 hybrids. MATERIALS AND METHOD Germplasm collection and initial evaluation Several accessions of germplasm were collected from open pollinated populations of local and introduced germplasm at farmer fields and few from foreign countries during the period of 2002-2006. Germplasm collection from farmer fields were done by collecting the fruits from hermaphrodite trees which were selected based on virus and disease free symptoms, more number of fruits and physical appearance of the fruits. Seeds of the exotic germplasm were collected when some of the scientists visited those countries. Fruits collected from farmer fields were brought to the lab and initially evaluated for external and internal quality characters such as fruit weight, fruit length, width, cavity size, peel thickness, flesh colour and taste. Based on these characters some accessions were selected for inbred line production. Development of inbred lines Twenty accessions were evaluated in the field at a time. Hundred seeds from each selected accession were sown in individual pots and 50 seedlings from each accession were planted in the field as progenies. Each individual in the progeny was assessed for growth, plant height, leaf type, and virus and disease incidence on visual basis. Flower type, number of fruits, fruit size and flesh colour were also recorded. Promising hermaphrodite trees were selected and selfed seeds were produced by bagging the flowers to raise the next generation. The same procedure was followed in subsequent generations until the population reached the sex ratio of 2 hermaphrodites: 1 female and uniform in fruit flesh colour and other desirable characters. Success in fruit setting in different types of hermaphrodite flowers Two hermaphrodite inbred lines developed in this study have elongated type flowers. For the maintenance of these two lines, selfing and sib mating were practiced and while doing so it was observed that at certain times of the season, the elongated flowers changed into petandria type flowers and most of these were found to drop without producing fruits (Fig. 1). Hence fruit setting in the two types of flowers under controlled (bagging the flowers) and natural pollination (without bagging) was studied. Flower type (elongated flowers and petandria type flowers) and pollination method (bagged for self pollination and non-bagged for open pollination) were tested in a 2 x 2 factorial experiment. During the period of flower change, trees of the inbred line, CP13-29 planted in 5 blocks were used for the study. Five trees from each flower type (elongated and petandria) were selected from each block. Two
DEVELOPMENT AND MAINTENANCE OF INBRED PAPAYA LINES 119
flower buds from each tree were tagged and one was bagged to allow self pollination and the other was not bagged. The bagging was done one day before opening of flowers. Fruit set was recorded 8 -10 days after flower opening and 1 month thereafter, and the results obtained for 1 month after flower opening were analysed as a factorial experiment. RESULTS AND DISCUSSION Germplasm collection and initial evaluation A total of 98 accessions of papaya were collected during 20022006. Of these, 92 were local accessions collected in the form of fruits from farmer fields in dry and wet zones of Sri Lanka (Table 1). Five accessions were collected in the form of seeds from Tailand, India and Hongkong. In one accession, in the form of plant was received from CIC company. Most of the local germplasm were inherent to the country and were available in the homegardens in the past. They were rarely found during the latter part of the collection (2005-2006) and seem to be replaced mostly by off-springs of the introduced Red lady variety. Accessions collected in the form of fruits were assessed for their quality characteristics but some fruits were found damaged during transport. Hence only the data of 83 and 57 samples were obtained for external and internal fruit characteristics respectively (Table 2). Table 1. Papaya germplasm collected during 2002-2005. District/Countr y Kalutara Colombo Gampaha Rathnapura Hambanthota Anuradhapura Kandy Mathale CIC Honkong India Thailand
No. of villages 12 4 4 4 3 1 1 1 -
No. of accessions 45 5 13 5 18 4 2 1 1 (Plants) 1 (Seeds) 1 (Seeds) 2 (Seeds)
Place of collection Home gardens Home gardens Home gardens Plant nursery and Home gardens Home gardens Home gardens Home gardens Home gardens -
Among the collected accessions, very few (8 accessions) had small sized fruits having 250-500 g/fruit while 40% had the medium sized fruits having 501-1000g/fruit and 46% had large fruits having >1000g/fruit. Majority of the accessions were yellow in fruit fresh colour while only 16% were in red and 19% were in orange to dark yellow (Table 2). Fruit size (length, width), flesh thickness, fruit cavity size, adherence of placenta to flesh and taste also varied among the accessions.
120 PERERA et al. Table 2. Fruit quality characteristics of collected accessions of papaya. Fruit weight (g)
Fruit length (cm)
Fruit width (cm)
Fruit cavity (cm)
Range
No. Acc . 8
Range
Range 5-10
No Acc . 35
Range
10-15
No. Acc . 5
33
15-20
37
11-15
38
2.1-3
6
2.1-3
32
DY
3
25
21-25
39
16-20
10
3.1-5
24
3.1-4
9
OR
8
17
26-30
2
0
5.1-7
19
0
R
9
7.1-9
7
250 500 501 1000 1001 1500 1501 2000 >2000 Total
>20
0 83
83
83
<2
No. Acc . 1
57
Flesh thickness (cm) Range No. Acc . 1-2 16
>4
Fruit colour
Y
57
No. Acc . 37
57
Y - Yellow, DY - Dark Yellow, OR - Orange, R â&#x20AC;&#x201C; Red, No. Acc. â&#x20AC;&#x201C; number of accessions
Fruit cavity size varied from <2 cm - 9 cm and of these, 54% were in less than 5cm while 33% were moderate having 5.1-7 cm and 13% were large. Flesh thickness varied among 1- 4cm and majority were (56%) in the range of 2.1-3 cm while 16% had flesh thickness of 3.1-4 cm. In developing papaya varieties, the breeding objectives in fruit quality characters are to produce small to medium size fruits with small ovarian cavity, large flesh thickness, easily separable placenta and good taste. Hence, the possible correlations among characters (fruit width, cavity width, flesh thickness, fruit weight and fruit width to cavity ratio) were computed in order to facilitate the selection criteria in subsequent generations in inbred line production (Table 3). Of these characters, fruit width and ovarian cavity width showed high positive correlation while fruit cavity and flesh thickness showed low positive correlation. It was observed that some accessions had small fruit cavity (1.6 cm) with small flesh thickness (1.5 cm) whereas some had large fruit cavity (8.3 cm) with fairly large flesh thickness (2.6 cm). This suggested that selecting fruits for small fruit cavity alone would not give large flesh thickness. Based on this initial information, 55 accessions having medium size fruits with large flesh thickness and medium to small ovarian cavity and good taste with other desirable characters were selected for inbred line production. Table 3. Correlation between different fruit characteristics. Variable Fruit width vs. Fruit cavity Fruit cavity vs. Flesh thickness Fruit weight vs. Fruit cavity Fruit weight vs. Flesh thickness Width:Cavity vs. Flesh thickness
R2 + 0.866 + 0.278 + 0.691 + 0.545 0.02
Production of inbred lines Fifty five selected accessions were raised in G 1 and their populations were studied. Out of these, all individuals in 6 populations were
DEVELOPMENT AND MAINTENANCE OF INBRED PAPAYA LINES 121
infected with viruses before flowering and another 28 populations at fruiting stage and they were removed. Of the remaining 21 populations, all were segregating into different ratios of sex forms of male, female and hermaphrodite trees. Two populations segregated for fruit flesh colour into red and yellow while 13 produced only yellow fleshed fruits, 5 produced red and 1 had only dark yellow flesh colour fruits. Five populations were selected on the basis of virus tolerance, growth and fruit characters for subsequent generation advancement (Table 4). In G2, another population was totally infected with virus before flowering and others had different degree of infection and subsequently only 2 populations were brought up to G 5. Segregating pattern of sex and fruit colour of these populations in G 3-G5 are presented in Table 5 and 6. Fruit flesh colour which was segregating to red and yellow in both populations reached uniformity in CP 13-29 in 3rd generation (G3) and in CP 1-2 in 4th generation (G4). Both populations had sex ratio of 2 hermaphrodites to 1 female in G4 and χ2 values computed for sex were lower than that of tabular values at 1% significant level. Both characters were stable in G5 (Table 7) showing that these lines reached uniformity and can be used as true-to-type inbreds/varieties (Fig. 2 and 3). Table 4. Segregation patterns of sex and colour in G2. Population
Total No. of plants
No. of flowering plants
Sex
CP1-2 CP2-3 CP13-3 CP13-29 CP13-31 - male
120 118 2 43 29 uprooted due to viruses 91 83 11 35 102 99 40 88 85 29 - female - hermaphrodite
No. of plants at bearing
Flesh colour
73
102
red and yellow
37 59 56
71 71 27
red red red
Table 5. Segregation patterns of sex and colour in G3. Population
CP1-2
Total No. of plant s 119
CP13-29
103
No. of flowering plants
Sex
102
0
36
73
0
23
No. of plant at bearing
Flesh colour
66
67
red and yellow
50
33
red
Table 6. Segregation pattern of sex and fruit flesh colour and the χ2 values computed for sex in G4. Populatio n
Total No. of plants
No. of flowerin g plants CP1-2 164 135 CP13-29 95 85 ns- at 1% level of significance
χ2
Sex
value df = 1 0 0
39 29
96 56
0.12ns 0.023ns
No. of plants at bearing 38 39
Fruit flesh colour yellow red
122 PERERA et al. Table 7. Segregation pattern of sex and fruit flesh colour and the Ď&#x2021;2 values computed for sex in G5. Population
Total No. of plants
No. of flowering plants
CP1-2 18* CP13-29 44 at 1% level of significance * - less due to hedge-hog damage
13 42
Ď&#x2021;2 value
Sex
df = 1 0 0
4 13
9 29
0.038ns 0.160 ns
Fruit flesh colour yellow red
Success in fruit setting in different types of hermaphrodite flowers At the beginning of bearing, both of these hermaphrodite lines had good number of fruit set but at certain times of the season, elongated flowers were found to change into petandria type of hermaphrodite flowers and male flowers. Change into petandria type was relatively more when males were very few (one or two). The flower type and bagging showed significant effect on success of fruit setting in papaya trees and the interaction was significant (Table 8). The elongated type flowers had significantly higher fruit set over the petandria flowers, both under bagging (controlled pollination) and without bagging (natural pollination). Most of the petandria flowers dropped when they were bagged and had very low success in fruit set (2%) over without bagging (40%). Fruit set in elongated flowers under bagging and without bagging was not significantly different showing that auto selfing was not affected due to bagging. However, under both conditions, elongated flowers had moderate level of fruit setting (72%). The elongated flower type found in both inbred lines was characterized by an elongated pistil with five stigmatic rays and five petals which were fused to about two third of their length, forming a corolla tube. There were ten stamens with short filaments attached to the throat of the corolla tube in two series of five below the stigmatic rays leaving around 4-5 mm space between stigmatic rays and anthers (Fig. 4). However, some stigmatic rays were found bending more towards stamens and touching anthers and this morphological arrangement would have allowed auto selfing resulting in moderately higher percent of fruit set in bagged flowers. However, the artificial pollination is skill required to have the highest level of fruit set in this form of elongated flowers. Table 8. Success of fruit set in petandria and elongated type of flowers under bagging and without bagging. Flower type
% Fruit setting Bagging Elongated 60.9* (76) Petandria 3.9 * (2) LSD (0.05) 19.1 CV% 34.9 * Transformed values based on arcsin transformation Percentages are given in the parenthesis
Without bagging 58.6* (68) 31.1* (40)
DEVELOPMENT AND MAINTENANCE OF INBRED PAPAYA LINES 123
Figure 1. Absence of fruits at the nodes where flowers changed into petandria type.
A B C
Figure 2. Characteristics of inbred line CP 13-29; bearing tree (A), whole fruit (B), internal appearance of the fruit (C).
A B C Figure 3. Characteristics of inbred line CP 1-2; bearing tree (A), whole fruit (B), internal appearance of the fruit (C).
1
124 PERERA et al.
DEVELOPMENT AND MAINTENANCE OF INBRED PAPAYA LINES 125
B A Figure 4. Hermaphrodite elongated flower type found in two inbred lines. A â&#x20AC;&#x201C; Un open, B - Open flower.
Figure 5. Perfect hermaphrodite flower type.
A
B
Figure 6. Hermaphrodite Petandria flower type found after change. A - Un open, B - Open flower
126 PERERA et al.
Another type of hermaphrodite flowers which were considered as perfect form were reported to be present in gynodioecious line in which the stamens were attached very close to the stigmatic rays and touching, well ensuring higher percent of automatic selfing (Fig. 5). This form of flowers was found in varieties of Red lady and Rathna. The petandria type flowers did not form a corolla tube and had only five stamens with relatively long filaments attached to the base of the ovary. However, the anthers lay far below the stigmatic rays (Fig. 6). This arrangement of anther and stigma resulted in poor fruit set in petandria flowers under both conditions, showing that the flowers need artificial selfing for fruit set and seed production. The exact reason for sex reversal in papaya is still not clear and it is suggested to be due to the genotype x environment interaction. Seasonal shift in sex form in hermaphrodite trees have been reported in some studies (Lange, 1961; Teotia and Singh, 1967; Veerannah, 1984) in which temperature, long summer days, high elevation, and water availability were discussed as some reasons for the cause. The perfect form of hermaphrodite flowers was found in the varieties of Red lady and Rathna; however, it is observed that they too occasionally had petandria flowers in very few numbers. Hence a detailed investigation is required to understand the seasonal effect on sex change as the productivity is likely to be affected. CONCLUSIONS The incidence of viruses was severe in Wet Zone (WZ) of the country and carrying out a long term breeding programme at a research station in WZ was very difficult and not producing an output to the expectation and to the effort taken unless we have resistance sources to work on. Two gynodioecious inbred lines were produced by selfing desirable hermaphrodites up to 4-5 generations and these can be used as parents in F1 hybrid production. When developing gynodioecious inbred lines selecting hermaphrodite trees having perfect form of elongated flowers and whose sex do not change with climatic variation needs attention. ACKNOWLEDGMENTS Authors wish to thank Ms. I. Kalubowila, Research Officer, for her involvement in germplasm collection and evaluation at the initial stage of the project and Ms. M. Jayawardana, Program Assistant for helping in the statistical analysis of the study. Council for Agricultural Research Policy is
1
DEVELOPMENT AND MAINTENANCE OF INBRED PAPAYA LINES 127
acknowledged for providing funds for the period of 2002-2007. Thanks also go to Ms. T. Diwakara, NAITA trainnee for type setting the manuscript.
128 PERERA et al.
REFERENCES Chan, Y.K. 1992. Progress in breeding of F1 papaya hybrids in Malaysia. Acta Horticulturae 292:41-49. Dinesh, M.R. and I.S. Yadav. 1998. Surya: a promising papaya hybrid. Indian Horticulture 43(3):21-33. Lange, A.H. 1961. Factors affecting sex changes in the flowers of Carica papaya L. American Society for Horticulture Science 77:252-264. Ram, M. 1982. Papaya improvement through selection and breeding technique. Punjab Journal of Horticulture 22:8-10. Ram, M., P. K. Majumder, B.N. Singh and A. Akgtar. 1999. Study on hybrid vigour in papaya (Carica papaya L.) varieties. Indian Journal of Horticulture 56(4):295-298. Singh, I.P. and C.K. Sharma. 1996. HPSC - 3: A high yielding new papaya hybrid for Tripura. Journal of Hill Research 9(1):73-75. Subramanyam, M.D. and C.P.A. Iyer. 1984. Exploitation of heterosis in papaya. Indian Journal of Horticulture 41:40-46. Storey, W.B. 1976. Papaya (Carica papaya). In Evolution of Crop Plants, Eds, N.W. Simmonds. Pp 21-24. Longman Inc. New York. Teotia, S.C. and R.N. Singh. 1967. Seasonal variation in sex expression of papaya ( Carica papaya L). Indian Agriculture 11:45-49. Veerannah, L. 1984. Seasonal rytham on the sex expression of papaya. In Proceedings of National Seminar on Papaya and Papain Producton. Pp i-iii. Tamil Nadu Agril. University of Coimbatore, Madras, March 26-27.