SELF INCOMPATIBILITY FINAL

SELF INCOMPATIBILITY IN VEGETABLES
Mode of pollination is very important in plant breeding because it determines the genetic constitution, nature of gene action, ease in pollination control and stability of varieties after release. There are several mechanisms that promote cross pollination, among these self-incompatibility. Self incompatibility is defined as the prevention of fusion of fertile (functional) male and female gametes of the same plant (Gowers, 1989). Self incompatibility is a system where self-recognition and rejection is the rule that prevents inbreeding depression.

1. By B. Pavan Kumar Naik 1 st Ph. D Horticulture Credit Seminar on SELF INCOMPATIBILITY IN VEGETABLES Annamalai University Faculty Of Agriculture Department Of Horticulture, Chidambaram Chairperson Dr. J. Sam Ruban Associate Professor Department of Horticulture Members 1. Dr. Arumugam Shakila Professor Department of Horticulture 2. Dr. Y. Anbuselvan Professor Department of Genetics & Plant Breeding

2. CONTENT Introduction Key breakthrough in timeline of research Main features of self-incompatibility Mechanism of SI Bateman classification of self-incompatibility Lewis classification of self-incompatibility Methods to asses self incompatibility Molecular model of SI in Brassicaceae Molecular model of SI in Solanaceae Need to use of self-incompatibility in the hybrid breeding Significance of self-incompatibility (Hybrid production) Temporary suppression of self incompatibility Limitations in exploiting self-incompatibility

3. INTRODUCTION First reported by Koelreuter (1850) Growers (1989) has defined self-incompatibility as “the prevention of fusion of fertile (functional) male and female gametes after self-pollination. Self-incompatibility is a genetic mechanism of avoiding self-fertilization that leads to natural out breeding where self-recognition and rejection is the rule. It promotes heterozgosity and prevents inbreeding depression.

4. Key breakthroughs in the timeline of research on Self-Incompatibility

5. MAIN FEATURES OF SI SI important outbreeding mechanism SI species do not produce seed on self pollination SI maintains high degree of heterozygosity & reduces homozygosity SI results due to morphological, genetic, physiological and biochemical causes

6. Bateman (1952) Classification of self-incompatibility : 1. Complementary systems of self-incompatibility 2. Oppositional system of self-incompatibility When pollen and pistil belong to same SI they produce certain chemicals which will prevent pollen germination and growth. In a compatible reactions no chemicals are inhibiting normal growth and development of pollen will be resulting in a successful fertilization. Pollen of one SI group Stigma of other SI group stimulate germination and growth of pollen tube results successful fertilization

7. Classification of Self-incompatibility Lewis, 1954

8. HETEROMORPHIC SYSTEM In the heteromorphic system, there are morphological differences among the flowers of the same plant. eg ., primula ( Primula species) there are two types of flowers pin ( ss ) having long style and short stigma and thrum having a short style and long stigma ( Ss ). This character is controlled by a single gene S; having two alleles. The alleles for short style are dominant over long style. The only compatible mating is between pin and thrum flowers and progeny have pin and thrum flowers in 1:1 ratio. CROSSES RESULTS Phenotype Genotype Genotype Phenotype Pin x Pin ss x ss Incompatible mating Pin x Thrum ss x Ss 1 Ss : 1 ss 1 Thrum: 1 Pin Thrum x Pin Ss x ss 1 Ss : 1 ss 1 Thrum : 1 Pin Thrum x Thrum Ss x Ss Incompatible mating

9. Conti… In some plant another situation called tristyly occurs. In this case style are of three length: Ex. Brinjal

10. HOMOMORPHIC SYSTEM S elf incompatibility results due to physiological causes rather than differences in flower morphology. In this system, the plants do not have differences in the length of style and stamens or other floral parts. This system is very much important in crop plants. It can operate in various ways, The pollen grains do not germinate on the stigma of same flower . If they germinate the pollen tube fails to penetrate the stigma as in Rye, Cabbage and Radish. The pollen grains may germinate but there is retardation of pollen tube growth. In some cases, there is slow rate of pollen tube growth and it rarely reaches the ovary in time to effect self fertilization.

11. Gametophytic system Incompatibility reaction of pollen is determined by its own genotype. S-allele in style show co-dominance

12. Sporophytic system In this system the self-incompatibility is governed by a single gene, S, with multiple alleles. Incompatibility reaction of pollen is governed by the genotype of plant, It was 1 st reported by Hughes and Babcock in 1950

13. Mechanism of SI Pollen-stigma interaction Pollen tube – style interaction Pollen tube – ovule interaction Dresselhausa and Franklin-Tong, 2013

14. https://getrevising.co.uk/revision-cards/plant-reproduction

15. METHODS TO ASSESS SELF INCOMPATIBILITY Pollination method Cytological method Molecular method Narayanapur et al. 2018

16. Pollination Method Pollination methods varies depending upon the type of self-incompatibility and crop. For example, in cabbage, after selfing , the self-incompatibility is assessed by counting the number of seeds after harvest. After selfing wait for 60 days till the pod maturity and count the number of seed set per pod. If more number of seeds are formed then it is self-compatible, if less seeds are formed then it is incompatible.

17. RESEARCH STUDY Ma, et,al . developed B. napus Self-incompatible line i.e , S-1300. Their result shown that salt solution can overcome the self-incompatible of S-1300 and this can be amplified by salt spraying with bee or hand pollination. Chaozhi Ma et al . 2009

18. Cytological Method In cabbage, the self-incompatibility reaction is assessed by number of pollen tube penetration in the style. Stigma and style are squashed on a microscope after 48 hours of pollination. Aniline blue is the stain used . It will get accumulated in the pollen tube and become fluorescent when irradiated with UV light . If there is no or less penetration of pollen tube in the style, then it is incompatible. If penetration of pollen tube is intermediate, then it is semi-compatible and penetration by many pollen tube in the style then it is compatible . Narayanapur et al. 2018

19. Assessment of self-incompatibility in cabbage by cytological method

20. FIG: Floral morphology and pollination behaviour during flower development in A. thaliana and A. halleri . Appearance and inner morphological structure of flower buds, with floral developmental stages (Smyth et al., 1990). Flower buds of Old-1 and W302 were emasculated at developmental stage 12 and incubated at 22°C on 1% agar medium until appropriate developmental stages. Each emasculated pistil at developmental stages 12, 13, 14, and 15 was self-pollinated in W302 and cross-pollinated with pollen of W302 in Old-1 to test the female SI function of Old-1. Scale bar, 1 mm. Self-incompatible and self-compatible floral stages are highlighted with blue and yellow boarders, respectively, in self-pollinated pistils of A. halleri and A. thaliana pistils pollinated with A. halleri pollens. Arrows indicate growing pollen tubes. Scale bar, 0.1 mm . ( Keita Suwabe et al. 2020) RESEARCH STUDY

21. Molecular basis of self-incompatibility Self-incompatibility system has been studied extensively in case of Solanaceae and brassicaceae family of vegetables . It involves two proteins located at S locus. One is male determinant and other is female determinant.

22. S. locus controlling self-incompatibility The S-locus complex and a model of self-incompatibility in Brassica . June B. Nasrallah , 1977

23. List of the identified female and male determinant genes Family Types of SI Male determinant Female determinant Brassicaceae SSI SP11/SCR SRK Solanaceae GSI SLF/SFB S- RNase Muthuselvi and Praneetha , 2019

24. S locus/ S related genes and their function/s in Brassicaceae members S-locus related gene Gene function Species name SLG Doubtful B. oleracea SRK Female determinant B. oleracea SP11/SCR Male determinant B. rapa MLPK Positive regulator B. rapa ARCI Positive regulator B. napus THLI Negative regulator B. napus KAPP Putative SRK interactor B. oleracea Calmodulin Putative SRK interactor B. oleracea sp locus Putative suprpressor B. napus Narayanapur and Minimol , 2018

25. The number of alleles and type of self-incompatibility identified in different plants Crop No. of S alleles Type of SI Reference B. oleraceavar . capitata 50 Sporophytic Bassett (1986) B. campestris 30 Sporophytic Singh (2012) Raphanus sativus 32 Sporophytic SI Karron et al. (1989) Raphanus raphanistrum 9 Sporophytic SI Sampson (1964)

26. Molecular model of SI in Brassicaceae Two genes SP11/ SCR is present, SP11 is male determinant and SRK is the female determinant. SLG is promoter of incompatibility reaction . SRK acts in the plasma membrane of papilla cells of stigma while SP11 get expressed in the anther tapetum during the maturation of pollen grain. In a SI reaction, when pollen grain land on stigma SP11 will bind with SRK and leads to autophosphorylation resulting in prevention of pollen tube growth. But in a compatible reaction, SP11 is not get activated and hence, normal pollen germination and fertilization takes place.

27. Vijayakumar et al. 2018

28. Molecular model of SI in Solanaceae In Solanaceae family, SLF/ SFB is the male determinant and S- RNase is the female determinant . During SIC reaction, when pollen falls on stigma, S- RNase is produced and enter into the stigmatic surface . They will degrade the RNA encoding the enzyme for pollen tube growth and result in death of pollen tube . In incompatible reaction also RNase is produced and it enter the stigma but it goes and forms a complex with SLF . Hence , RNA encoding enzyme for pollen tube growth is not disturbed and results in normal pollen tube growth and fertilization.

29. Significance of self-incompatibility Self-incompatibility effectively prevents self pollination . As a result, it has profound effect on breeding approaches and objectives.

30. Hybrid production Self-incompatibility is primarily exploited for hybrid production. If self-incompatible lines are available tedious process of emasculation can be avoided. If both lines are self-incompatible, they can be utilized for forward and reverse crosses. By using self-incompatibility double cross and three way cross hybrids can be produced.

31. Single, Double and three way cross hybrid seed production FIG: Application of self-incompatibility in practical plant breeding. Sporophytic incompatibility is widely used in breeding of cabbage and other Brassica species. Single-cross hybrids are more uniform and easier to produce. The top cross is commonly used. A single self-incompatible parent is used as female, and is open pollinated by a desirable cultivar as the pollen source . ( Gulzar et al. 2012)

32. RESEARCH STUDY Chaozhi Ma et al. 2009 a b c d Visualization of seed set of self-incompatible line S-1300 in different conditions a : selfing in isolation; b :selfing and bud-pollinating; c : propagation by salt solution; d : hybrid seed production

33. RESEARCH STUDY M. NIEUWHOF, 1974 From 30 varieties and strains of cauliflower the degree of self-incompatibility was tested. It was found that the early cauliflower varieties show a low level of self-incompatibility and most of the late cauliflower varieties a higher level of self-incompatibility .

34. Conti… The different reaction of the self-compatible and the more self-incompatible varieties is also reflected after bud-pollination. When bud-pollinating the first group of varieties seed-set was mostly lower than when selfing freshly opened flowers, on an average 19 %. This indicates that when no incompatibility barrier is present the conditions for seed production are more favourable after selfing freshly opened flowers than after selfing the young buds. But when such a barrier is present, as in self-incompatible varieties, bud-pollination is more effective. Thus , the average seed-set of the self-incompatible varieties was 89% higher after bud-pollination than after selfing open flowers. M. NIEUWHOF, 1974

35. Conti… It was clearly demonstrated that early cauliflower varieties are very self-compatible . After selfing the early varieties observed a somewhat lower seed set than after crossing. This might indicate the presence of a low degree of self-incompatibility in this group of varieties . The autumn and winter varieties possess a distinctly higher degree of self-incompatibility than the early cauliflower varieties. M. NIEUWHOF, 1974

36. RESEARCH STUDY P. K. SINGH, 2012

37. Conti… P. K. SINGH, 2012

38. Conti… Hybrid seed production using self-incompatibility involves the development and maintenance of parents which are homozygously selfincompatible but cross-compatible, so the degree of self-incompatibility and stability in the inbred lines is of utmost importance. This is largely dependent on environmental factors, especially temperature. Uniform , effective cross-pollination may be of great importance for obtaining a high quantity of hybrid seed in self-incompatible types . The two inbred lines, Desi Red and Khasi Kata, can be used as parental lines to produce single cross hybrid seed. The lines should be grown in alternate rows or with two to four rows of each, and adequate insect pollination should be ensured. The F1 populations of such hybrids will be uniform. P. K. SINGH, 2012

39. RESEARCH STUDY SI has been reported to be preferred over male sterility in crop species with entomophilous pollination since pollen-collecting bees rarely visit male-sterile plants. Nonetheless , SI also may have disadvantages, Ex. F1 hybrids of two SI parents are also SI and this may be undesirable for crop production. SI F1 hybrids are not a handicap for ornamental or vegetable crops, but it may hinder seed production (e.g., oilseed rape). Consequently , breeding programs favor not only SI female lines, but also SC F1 hybrids ( Kaothien -Nakayama et al. , 2010) Muñoz- Sanz JV et al. 2020

40. SI based systems as alternatives to androsterility for producing F1 hybrids. Self-(in)compatible parents and F1 hybrids are indicated.

41. Hybrids developed using self-incompatibility CROPS HYBRIDS Cauliflower Pusa Hybrid 2, Pusa Kartik Sankar , Snow queen, Snow king Cabbage Pusa Synthetic, Pusa Cabbage 1, Meenakshi , BRH-5, H-43, H-44 Chinese cabbage Hamburg-3 Raddish Pusa Chetaki , Pusa Desi , Half Red, Chinese Pink, BDI-689

42. Need to use of self-incompatibility in the hybrid breeding of vegetable crops Used in hybrid seed production . Self incompatible line may be inter planted with a self compatible line. Seed from only the self-incompatible line would be hybrid.  The production of double cross and triple cross hybrids can be produced in case of Brassicas .

43. Level of SI in Brassica oleracea L. Crop Level of SI Reference Kale and Round headed cabbage High Thompson, 1965 Nelson, 1927 Broccoli High to moderate Nelson, 1927 Thompson and Taylor, 1965 Autumn and winter cauliflower Moderate Watts, 1963 White sprouting broccoli and brussels -sprout Moderate to low Johnson, 1955 Green kohl rabi and purple sprouting broccoli Low Nelson, 1927 Early summer cauliflower Very low Watts, 1963,

44. TEMPORARY SUPPRESSION OF SELF INCOMPATIBILITY Bud Pollination Increased CO2 concentration Salt ( NaCl ) sprays High temperature treatment

45. RESEARCH STUDY BUD POLLINATIONS

46. RESEARCH STUDY -Cabin et al. 1995

47. RESEARCH STUDY SALT ( NaCl ) SPRAYS

48. - Carafa and Carratu , 1997 RESEARCH STUDY

49. Conti… Self pollen grain germinate and pollen tubes penetrate the stigmatic papillae after stigma treatment with 0.5 M NaCl .

50. Conti…

51. RESEARCH STUDY INCREASED CO 2 CONCENTRATION

52. - Dhaliwal and Malik, 1982 RESEARCH STUDY

53. Conti…

54. RESEARCH STUDY SHORT-TERM HIGH TEMPERATURE TREATMENT

55. Okazaki and Hinata , 1987 RESEARCH STUDY

56. Conti… Many pollen tubes penetrated the pistil in self pollination after the 40 o c - 15 min treatment (x 100) Callose formed in papillae (arrows) in which selfed pollen tubes had penetrated after treatment (x 200 ). Abnormal pollen tubes growing in pistil treated at 50 o C for 15 min ( x 200)

57. Limitations in exploiting self-incompatibility Continues selfing will lead to inbreeding depression. Continues inbreeding may lead to complete loss of the inbred lines . Hybrid seed would be expensive if the self-incompatible lines are difficult to maintain. Environmental factor reduce or totally overcome SI. Preferential visit of pollinating insects. Transfer of S-allele is tedious and complicated.

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