Advances in dwarfism of fruit plants

Physiology of dwarfism and methods to achieve dwarfisms

1. Welcome

2. Master Seminar Advances in dwarfism of fruit plants Warang Omkar Sunil M.Sc (Hort.) Fruit Science Course Instructor Dr. B. R. Salvi Chairman & Research guide Dr. P. M. Haldankar Department of Horticulture, College of Agriculture, Dapoli

3. Content Introduction Principle Physiology of dwarfism Methods to achieve dwarfism Research findings Thrust areas Conclusion

4. Introduction Dwarfing is an alteration in the normal growth pattern. A dwarf plant is that which is smaller than normal size at full maturity and possess other characteristics like precocity, canopy architecture and time of flowering and altered fruit size.

5. Principle To make the best use of vertical and horizontal space per unit time and to harness maximum possible returns per unit of inputs and natural resources.

6. Physiology of dwarfism Mechanism - involves anatomical, physiological and biochemical changes . Auxins are produced by shoot tips and translocated basipetaly downwards to the roots through phloem. Dwarfing rootstock controls tree Size -by controlling the auxin passing through the bark of the rootstock . large number of vessels and twice as many xylem fibers are produced in vigorous rootstocks than dwarfing ones ( Beakbane et al., 1974 ). A greater number of non-functioning phloem has been found in rootstock with thick bark (M9) than those with thin bark i.e MM106 or M7 ( Chu and Simons, 1984).

7. Methods to achieve dwarfism Dwarfness can be achieved by various ways such as:- Use of dwarfing root stock/ interstock or use of dwarfing scion Use of bioregulators Use of incompatible root stock Induction of viral infection Pruning and training Nutrients Phenols 8. In vitro techniques Genetic engineering Ringing or girdling Others

8. Use of dwarfing root stock/ interstock U se of vigour controlling root stocks is one method used to promote early fruit bearing, reduce vigour and increased yield. Various studies suggested a correlation between early flowering and smaller tree size ( Costes and Garcia, 2007). Apple dwarfing rootstocks induce two most important effects i.e precocity and reduction in tree size ( Lauri et al ., 2006).

9. Correlations for predicting dwarfing capacity of rootstocks Percentage of live tissues of root cross section. Bark : wood ratio Percentage of ray tissues in root cross section High stomatal density on leaves Bark is the key factor as it reduces translocation of auxins , sugars and other compounds. Genes exert their effect on dwarfness .

10. Physiological processes of root stock inducing dwarfness Anatomy of Dwarfing rootstock:- Smaller xylem vessels and less xylem fibers Have a higher percentage of living tissues than lignified cells Higher percentage of bark and wood ray tissues 2. Nutrition:- More carbohydrates are directed to fruiting structure and less to tree frame by dwarfing rootstock.

11. 3. Hydraulic conductivity:- Reduced root hydraulic conductance helps to induce dwarfism ( Nardini et al; 2006 ). 4. Translocation of water and minerals:- Less effective in uptake of nutrients and water Partial blockage at graft union affects water and nutrients translocation e.g. Reduction in translocation of P and Ca ( Bukovac et al . , 1958).

12. Phytohormones :- Less auxin flow take place through bark of dwarf rootstock Lower concentration of IAA and ABA are observed in dwarfing rootstock while vigorous rootstock showed higher cytokinin concentration in root pressure exudate and shoot xylem sap. Lack of ability to produce soluble sugars result in reduced growth e.g starch hydrolysis. NAA suppress the conversion while IAA promotes it that is why NAA cause dwarfing.

13. 6. Bark of the rootstock:- Thicker the bark lesser the height as auxins get destroyed by several enzymes. e.g.- dwarfed avocado trees had 22.7 % of bark compared to 12.9 % in tall ones. Greater thickness in the bark of dwarf trees is associated with a higher degradation of auxins by IIA oxidase, peroxidase and phenolic compounds present in the bark ( Lockhard and Schneider, 1981).

14. Reduced root system of dwarfing rootstocks:- dwarf rootstocks have small and limited root systems. More dwarfing a rootstock the smaller is its root system. e.g.- M9 has limited root system compared to seedling rootstock (Fernandez et al ., 1995). Depletion of solutes in xylem sap of scions by dwarfing rootstock:- It has been observed that dwarfing root stocks and inter stocks lead to depletion of xylem sap with respect to N, P, K, Ca and Mg (Jones 1976).

15. Dwarfing root stocks induce restricted canopy development of scion variety:- Induce wider crotch angles to lateral branches and spreading habit in scions. Horizontal branches show both decreased vegetative growth and enhanced flowering and fruiting (Crabbe, 1984). Reduce the growth of both vertical and horizontal branches and effects being greater on vertical branches (Webster, 1995).

16. 10. Precocious and heavy fruiting at the expense of vegetative growth:- Trees on dwarfing root stocks show a higher ratio of fruit yield to vegetative growth than those on vigorous root stocks . 11. Reduced carbohydrates transport from leaves to root:- Dwarfing rootstock have been found to block the transport of carbohydrates from leaves to roots.

17. Some important dwarfing rootstocks of fruit crops 1. Apple :- M27, M9, EMLA 26. 2. Pear:- Quince C 3. Peach:- Siberian C, St. Julien X, P. besseyi and Rubira . 4. Plum:- St. Julien A, St. Julien K, Pixy. 5. Cherry:- Giesela series, Stockton Morello , Oppenheim, Charger. 6 . Mango:- Olour , Vellaikolumban 7 . Citrus:- Trifoliate Orange, Flying Dragon 8. Guava:- Pusa Srijan , P. friedrichesthalianum 9. Ber :- Jhar ber

18. Use of bioregulators A wide varieties of growth regulators re being used to regulate the growth rate of the fruit trees. Paclobutrazol :- It inhibit the GA synthesis and thereby retards growth, shorten elongation and reduced internodal length. Dwarfing mechanism due to PBZ is based upon:- i . Shorter internodes due to less GA in tissues ii. Reduces ABA levels in shoot tips (Kurian et al ; 1993). iii. Reduces the levels of cytokinins (Kurian et al ; 1993). iv. Enhances the total phenolic content of terminal buds (Kurian et al ; 1993).

19. Gibberlins biosynthesis inhibitors – Onium compounds - CCC, Mepiquat chloride, Phosphon -D. Triazoles - Uniconazole , Tripenthenol , Paclobutrazol . Pyrimidines - Inhibit synthesis of gibberlins . New molecules - Tetcyclasis , morphactins

20. Bioregulator Concentration Crop Effect Recommended by - PBZ 15 mg / liter Plum, pear, apricot Reduced shoot length Grochowska and Hodun (1997) TIBA 3.5 mg / liter Plum, sour cherry Reduced shoot length Grochowska and Hodun (1997) PBZ 2.5 – 5 g/l Mango Reduced 50% of tree volume Iyer and Kurian (1993) PBZ 500-1000 ppm Satsuma mandarin Inhibits vegetative growth Okuda et al. (1996)

21. Use of incompatible rootstock Dawrfness can be imparted by the use of incompatible scion and rootstock. Crop name Root stock Scientist Ber Jhar Verma et al., 2000 Pear Quince Francesscatto et al.,2007

22. Induction of viral infection Inducing viral infection It is not commercially adopted. Eg . Citrus, Apple. Symptoms of Citrus Exocortis viriods on Citrus plants

23. Pruning and training Slow growing trees responds more to pruning. A compact and bushy tree achieved by removal of apical portion . Limitation :- grape , apple and some other temperate crops . Of the various training systems being followed in apple e.g. spindle bush and single vertical axis raised on M9, M7 and M4 root stocks has been found promising for HDP w.r.t size control and higher yield per unit land area ( Gyuro , 1978). Spindle Bush system Vertical axis system

24. Nutrients Moderately vigorous trees utilize less nitrogen Vigorous one use more nitrogen.

25. Phenols Phenols reduce vegetative growth by inhibiting mitosis, cell division, cell elongation and increase IAA oxidation. Some phenols also inhibit translocation of sugar and auxin or act by regulating polar auxin transport. Eg . Coumarin , phloridzin

26. In vitro techniques Seeds are subjected to different level of BA (Benzyl adenine) and TDZ ( Thidiazuron ). The plants produced with TDZ and BA were slower in growth and dwarf in size than non-treated plants ( Khattak et al ., 2004).

27. Genetic engineering DNA coding of sorbitol- 6 –phosphate dehydrogense (S6PDH) which is the key enzyme in sorbitol biosynthesis introduced into Japanese persimmon induced dwarfism. The physiological mechanism behind this dwarfing effect has been attributed to accumulation of sorbitol which might have caused an osmotic imbalance between cytosol and vacuole ( Deguchi et al., 2004). Various genes have been identified in fruit crops that can induce dwarfism eg . Ipt ( isopentenyl transferase ), OSHI, rolA etc. In apple reduction in stem length and node number was achieved through over expression of gaai gene isolated from Arabidopsis (Zhu et al ., 2008).

28. Ringing or Girdling With the removal of bark the flow of carbohydrates down to the roots restricted and, therefore carbohydrates accumulate above the girdle producing differential effect on root development . This in turn, leads to differential effect on shoot growth . Cutting and Lyne (1993), supported the hypothesis that less cytokinins and gibberellins above the girdle are likely to reduce meristematic activity and cell elongation, leading to reduction in vegetative growth.

29. Others Proteins, tengu - su inducer (TENGU) excreted by bacterial phytoplasm inhibits auxin related pathways there by affecting plant development ( Hoshia et al., 2009). Down regulation of GA synthesizing genes. Dwarfing through mutations:- Gamma irradiation applied twice induced dwarfism in Irwin cv. of mango seedlings and the survival percentage was 65 % ( Yitzu and Loonshung , 2000 ).

30. Research Findings

31. Table no. 1. Tree growth of four apple cultivars on several rootstocks at 12 year old Cultivar Rootstock Tree girth Tree Crown Height Spread Jonathan M7 40. 5 4.4 4. 8 M9 23.2 2. 6 2. 8 M26 34. 4 3. 6 3. 8 MM106 46. 7 4. 6 5. 6 M. prunifolia 53. 0 4. 9 6. 5 Starking Delicious M7 40. 5 4. 4 5. 0 M9 25. 2 3.5 3. 6 M26 44.5 4. 9 5. 4 MM106 48. 3 5. 4 6. 5 M. prunifolia 58. 8 4. 7 7. 0 Golden Delicious M7 47.3 5.0 5. 8 M9 29. 3 3. 4 3. 4 M26 43. 0 4. 6 4. 8 MM106 54. 3 4. 9 6. l M. prunifolia 59. 3 4. 8 6. 6 Fuji M9 26.7 2. 8 3. 2 M26 53. 0 4.7 6.0 MM106 59.0 4. 6 6.0 M. prunifolia 64. 8 5. 0 7.2 Shichiro Tsuchiya (1979) Morioka, Japan Dwarfing Rootstocks of Apple

32. treatments Pooled mean of height (m) Plant volume (m 3 ) Pooled over the years mean(m 3 ) % reduction in volume 2003 2004 2005 2006 Ratna/ vellaikolumban 3.6 289.0 293.3 270.6 320.7 285.9 24.9 Ratna/ Mixed rootstock 3.7 360.8 334.5 329.5 498.0 380.8 Alphonso/ Vellaikolumban 3.8 306.1 303.1 270.7 414.2 323.5 39.1 Alphonso/ Mixed rootstock 4.7 582.6 465.4 483.3 593.9 531.3 Kesar/ Vellaikolumban 4.6 512.4 397.2 406.1 599.5 478.8 26.4 Kesar/ Mixed rootstock 4.6 679.5 569.0 576.0 781.4 651.5 SE± 0.17 57.3 48.2 50.0 76.5 27.9 C.D (p=0.05) 0.5 169.0 142.2 147.4 225.7 78.4 Table:2-comparative performance of mango varieties grafted on vellaikolumban and mixed rootstock (11 year old trees) Gawankar et al. (2010), Dapoli

33. Table no. 3- Effect of rootstocks on growth and vigour of new apple varieties Variety Plant girth (cm) Plant height (cm) Plant spread (cm) M-9 MM-106 M-9 MM-106 M-9 MM-106 Lal Ambri 17.99 17.44 194.14 216.30 178.97 172.18 Sunheri 16.99 17.08 195.91 212.92 171.62 168.05 Shireen 14.90 15.91 196.32 208.09 172.15 166.07 Fidrous 15.01 13.75 193.04 204.68 168.90 163.34 Akbar 15.90 14.82 192.24 209.33 170.77 168.81 CD @ 5% Rootstock 0.48 0.51 0.29 Variety 0.76 0.81 0.47 Rootstock x Variety 1.08 1.14 0.66 Rift et al., 2008, Kashmir

34. Rootstock Origin CA (m 2 ) Growth rate(cm 2 TCA/month) Vellaikulumban Sri Lanka 4.16 4.91 Chandrakaran Cochin, India 4.54 4.76 Saigon unknown 4.62 5.42 Pico Philippines 5.52 5.34 Elephant tusk Thailand 6.38 6.70 Phoenix Australia 6.67 7.51 Neil Australia 6.83 5.63 kurukan India/Sri Lanka 7.52 7.42 caraboa Philippines 6.54 6.45 Orange Indonesia 7.12 10.33 LSD 0.05 0.19 0.51 Table no. 4: Field evaluation of different rootstocks for growth of ‘Kensington Pride’ mango (4 year old trees) Canopy Area (CA), trunk cross sectional area (TCA) Smith et al., 2008, Australia

35. Table no. 5. Effect of different rootstocks on growth parameters of the Ber (cv. Seb ) plants during 1993-94 (5 year old plants) Rootstocks Plant height (m) Spread of plant (m) Trunk diameter (cm) E W N S Below union Above union Z.nummularia 1.85 2.0 2.40 3.7 5.2 Z. spinachristi 2.05 2.6 2.75 6.1 6.5 Z. mauritiana 2.20 2.9 3.05 7.0 7.3 Z. rotundifolia 2.34 3.4 3.50 7.4 7.8 CD at 5% 0.46 0.31 0.23 2.01 1.32 Prasad & Bankar (2006), Jodhpur, Rajasthan

36. Table no. 6:- Effects of interstock type, interstock length and grafting point height on vegetative growth of apple ‘‘ Annurca Rossa del Sud ’’ cultivar. Treatments Circumference of rootstock (cm) Circumference of scion (cm) Plant height (cm) Canopy Volume (m³) Graft combinations CV/M27/SR 21.9 19.9 280.2 8.9 CV/M9/SR 29.4 26.2 356.1 21.7 CV/SR 55.4 45.9 483.5 45.8 Interstock length 10 cm 28.9 25.1 342.7 19.3 20 cm 22.4 21.0 293.6 11.2 Grafting point height 10 cm 33.5 28.2 351.5 21.1 20 cm 29.7 21.7 351.0 21.7 Di Vaio et al. (2008), South Italy

37. Table no. 7- Rootstock/ interstock effects on number of different axillary annual shoot types grown by Royal Gala apple trees in their second year of development (8 year 0ld trees) Treatment Vegetative extension shoots Vegetative spurs MM.106 21.7±1.0 a 18.2± a MM. 106/MM.106 18.3±1.5 ab 16.4± ab MM. 106/M.9 13.5± 1.9 b 16.4±ab M. 9/MM.106 6.2± 1.3 c 10.6± b M. 9 5.8± o.7 c 11.7± b M. 9/M.9 6.6± 1.o c 9.9± 2.2 b Seleznyova et al ., 2008, New Zealand

38. Table no. 8- Irrigation water uptake in fully grown Golden Delicious apple trees on semi dwarfing MM 106, local Hashabi and dwarfing M9 rootstocks Rootstock Month Monthly total (mm) Sap flow Hashabi June 189 July 208 August 208 Total 606 M9 June 155 July 163 August 158 Total 476 MM106 June 225 July 227 August 231 Total 682 Cohen & Naor., 2002, Israel

39. Table no. 9:- Total soluble sugars concentration (% dry mass) in the scion, graft union, rootstock and rootstock shank of ‘Rainier’ sweet cherry trees on ‘ Gi 5’ and ‘Colt’ rootstocks   Rainier X Gi5 Rainier X Colt Scion 2.71 1.58 Graft union 1.92 2.23 Rootstock 1.44 2.39 Rootstock shank 1.66 2.35 ‘ Gi 5’ ( Prunus cerasus L. X Prunus canescens L., dwarfing) ‘Colt’ ( Prunus avium X Prunus pseudocerasus L., vigorous) M.A . Olmstead et al. (2010), East Lansing, Mich .

40. Table no. 10:- TCA, hydraulic conductance of roots and entire tree of Crimson Lady peach trees on Nemagaurd and KP-146-144 rootstocks Rootstock Trunk cross sectional area (cm 2 ) Hydraulic conductance of roots Hydraulic conductance of entire tree Nemagaurd 4.05 6.56 X 10 -5 AL 5.23 X 10 -5 AL KP-146-144 0.90 2.49 X 10 -5 AL 2.29 X 10 -5 AL Basile et al. (2003), USA Nemagaurd :- vigorous rootstock KP-146-144:- Dwarf rootstock

41. Table no. 11:- Mean comparisons of canopy size, height and yield per canopy volume of Commune clementine grafted on Pomeroy trifoliate orange infected with a single viroid Treatment Canopy volume (m 3 ) Height (m) Annual yield (kg/m 3 ) Viroid Isolate Control   20.68 3.40 3.91 CEVd CEVd-117 15.55 3.09 2.72   CEVd-129 15.21 3.20 3.06 CBL Vd CVd-Ia-117 18.64 3.26 3.60   CVd-Ib 18.83 3.29 4.12 HSVd CVd-IIa-117 20.30 3.31 4.51   CVd-IIb 19.45 3.33 3.48   CVd-IIc 17.35 3.21 3.49 CVd-III CVd-IIIa 15.29 3.05 3.29   CVd-IIIb 14.51 3.02 4.74   CVd-IIIc 12.69 2.88 4.93   CVd-IIId 12.62 2.77 3.61 CVd -IV CVd-IV-Ca 22.85 3.35 3.71 Vernière et al. (2004), France

42. Cultivars Plant height (cm) Leaf no. Stem girth (cm) Phenols (mg/g.) ABA (mg/g.) IAA (mg/g.) Bappakai 39.6 17.6 3.30 9.46 10.60 8.46 Muvandan 42.4 24.2 3.20 22.46 13.68 15.48 Olour 31.0 24.0 3.04 31.92 22.40 8.98 Mylepelian 33.6 17.2 2.80 14.78 20.81 8.98 Chandrakiran 23.0 9.4 2.20 39.60 26.94 11.09 Kurukan 25.1 13.2 1.80 50.24 28.97 7.90 Vellaikolamban 17.0 7.4 1.80 59.10 43.19 11.42 CD 5% 3.51 4.75 0.11 4.96 5.69 3.41 SEM 1.31 2.26 0.05 2.41 3.04 1.63 Table no. 12:- Endogenous Hormones and Phenols in Rootstock Seedlings of Mango Cultivars and their Relationship with Seedling Vigour Murti and upreti (2003), Banglore

43. Name of the cultivar BA conc. (µM) Shoot length (cm) No. of roots Root length (cm) Survival (%) Golden Delicious 8.1 7.3 1.8 100 20 4.8 4.6 3.6 60 40 3.9 2.6 2.6 30 60 2.7 2.0 1.5 3 USDA 4-20 7.9 7.5 1.9 100 20 4.5 3.6 3.0 50 40 3.0 2.7 2.7 20 60 1.0 1.0 1.3 2 LIberty 8.0 7.8 1.8 100 20 3.5 3.0 2.8 50 40 2.1 1.6 1.8 25 60 Table no. 13:- Growth and survival percentage of Ex-in vitro apple cultivars plantlets in green house (6 weeks studies) Khattak et al. (2007), Peshawar, Pakistan

44. Table no. 14:- Mean trunk cross-sectional area (TCA) and tree canopy volume for cv. Jonagold /M.9 apple trees in different training systems (5 years) Training system TCA (cm 2 ) Canopy volume (m 3 ) Slender Spindle 9.41 1.7 Verticle Axis 11.96 3.0 Hytec 14.85 4.13 Low-Super Spindle 8.74 2.50 High-Super spindle 7.17 1.45 SS – (4,761 trees/ha) VA – (2,857 trees/ha) HT – (1,904 trees/ha) L-Super S – (5,000 trees/ha) H-Super S – (10,000/trees/ha) Ozkan et al. (2012), Turkey

45. Table no.15:- Effect of the training system on trunk cross-sectional area, the canopy volume and crop load in the apple cultivar ‘ Braeburn ’ grafted on M9 rootstock (5 years) Training System TCA (cm 2 ) Canopy volume (m 3 ) Crop load (Number of fruit of cm²/TCA) Slender Spindle 30.2 2.33 2.39 Solen 24.6 1.80 2.24 Verticle Axis 31.6 3.81 3.07 Gandev et al. (2016), Bulgeria

46. Table no. 16:- Comparison of trunk cross ‑ sectional area, crown volume and yield efficiency per TCSA among training systems of ‘Topaz’ apple trees after 5 years. Treatment TCSA (cm 2 ) Crown volume (m 3 ) Yield efficiency per TCSA (kg.cm−2) SS-WP 26.70 3.161 2.813 SS-WP+SP 29.26 3.590 2.661 MS-WP 29.23 2.924 2.461 MS-WP+SP 29.25 2.908 2.423 TCSA = trunk cross sectional area CV = crown volume SS = slender spindle WP = winter pruning SP = summer pruning MS = modified slender spindle Sus et al. (2017), Czech Republic

47. Table no. 17:- Influence of training system on tree height, trunk cross sectional area, canopy volume and yield efficiency of Ziraat sweet cherry (4 years). Training system Tree height (cm) TCSA (cm 2 ) Canopy volume (m 3 ) Yield efficiency (kg cm¯² ) SB 243.4 44.65 2.59 0.32 SL 254.6 49.80 3.18 0.28 VCL 257.2 38.76 3.06 0.39 S.B: Spanish bush S.L.: Steep leader V.C.L.: Vogel central leader Aglar et al. (2016), Turkey.

48. Table no. 18:- Effects of HDP and two training methods on ‘ Jonica ’ dwarf apple trees grown in Sub Carpathian region (8 years old tree) Rootstock Training system Trees/ha Trunk cross sectional area(cm 2 ) M. 9 Slender spindle 2700 34.94 a 2050 30.21 a Vertical axis 3834 20.29 b 2700 24.94 b P 22 Slender spindle 2700 14.68 c 2052 15.87 c Vertical axis 3834 10.81 c 2700 13.38 c Adam and Augustyn , 2003, Poland

49. Table:19- Effects of methods and rates of paclobutrazol on tree height, volume, and shoot length of 'Tommy Atkins' mango 3 months after treatment application. Treatments Height of trees (m) Tree volume (m 3 ) Length of new shoots (cm) Soil drench 0 (control) 5.64 a 98.55 a 26.50 a 2.75 g a.i/ tree 5.24 b 90.06 b 23.09 b 5.50 g a.i/ tree 5.31 ab 90.07 b 23.24 b 8.25 g a.i/ tree 5.22 b 86.53 bc 22.99 b Foliar application 0 (control) 5.62 a 95.99 a 26.02 a 2.75 g a.i/ tree 5.30 ab 89.96 b 23.16 b 5.50 g a.i/ tree 5.30 ab 87.85 bc 23.13 b 8.25 g a.i/ tree 5.19 b 85.78 c 22.96 b SED 0.05 1,65 0.64 Teferi et al., 2004, New Zealand

50. Thrust Areas Understanding the physiology of dwarfing rootstocks and their effect on scion vigour . Commercializing the use of incompatible rootstocks and use of viral infections for dwarfing Need to develop dwarf rootstocks for evergreen subtropical fruit crops for HDP. Need to develop dwarf rootstocks which does not affect the fruit quality of scion cultivar. Opportunities will also exist to modify the root growth of crops for which no dwarfing rootstocks exist presently. Screening of dwarf cultivars for dwarfing genes in fruit plants eg . S6PDH, GID1c from brachytic dwarf peach.

51. Conclusion Dwarfing in fruit crops can be achieved through various approaches like use of dwarfing rootstock, root pruning, training, use of growth retardants, control of nutrient elements etc. P aclobutrazol are most effective and widely used growth retardant. Dwarfing rootstock plays important role in controlling tree size and induce precocious bearing in fruit plants . Pruning practices like tip pruning, pinching, removal of apical buds, heading back, etc. control the tree size. Ringing and girdling control the tree size by restricting the flow of carbohydrates and auxins to the roots. Recently genetic engineering has widen the gene pool which can be manipulated to induce dwarfism and harvest maximum benefits in horticultural crops.

52. Thank You