Novel technique of plant regeneration.

1. Welcome

2. Micrografting in Citrus Species
Department of Horticulture,
Rajasthan College of Agriculture, MPUAT,
Udaipur (Rajasthan)
Presented By
Saniya
Ph.D. Horticulture

3. Seminar Outlines
• Introduction
• Need of Micrografting
• Principle of Micrografting
• Applications of Micrografting
• Procedure of Micrografting
• Advantages
• Limitations
• Affecting factors
• Case studies
• Conclusion

4. How to deal with citrus graft-transmissible
diseases which results in serious economic
losses in most citrus growing
regions?

5. Solution
Micrografting
or
Shoot tip grafting

6. What is micrografting/shoot tip grafting?
“Micrografting is an in vitro
grafting technique which
involves the placement of a
meristem or shoot tip explant
onto a decapitated rootstock
that has been grown
aseptically from seed or
micropropagated cultures.”

7. One of the most ingenious grafting innovations of the 20th
century, Murashige et al. (1972) were able to recover a few citrus
plants by grafting shoot tips from diseased plants on young
rootstock seedlings growing in vitro. Some of these plants were
free of the exocortis viroid and did not have juvenile characters.
This procedure was studied in detail by Navarro et al. (1975),
who named it shoot tip grafting in vitro (STG), and developed a
routine procedure that allowed a 30-50% incidence of successful
grafts that were transplanted to soil, with over 95% survival rate.
Introduction

8. Why it is needed?
Pathogen-free plants of many cultivars are often not
available and it is necessary to recover healthy plants
from infected ones. In this situation, a method able to
recover citrus plant free of all graft-transmissible
pathogens and without juvenile characters is required to
produce healthy trees for commercial propagation.

9. Shoot tip grafting is simply based on the principle that
virus pathogens do not always invade newly developing cell
tissue as it is rapidly dividing and expanding. These small
shoot tips do not have vascular connection with the rest of
the plant.
Principle of successful micrografting

10. Concept of micrografting
The technique exploits two concepts:
(1) meristems are relatively virus and
pathogen free; and
(2) meristems from mature plants retain
the mature phase. Thus, the use of in
vitro shoot tip grafting produces plants
that are virus free and reproductively
mature.

11. Applications of Micrografting
Virus and Viroid Elimination
Assessment of Graft Incompatibility
Improvement of Plant Regeneration
Mass Multiplication
Indexing Viral Diseases
Safe germplasm exchange
Application of micrografting

12. The technique has the following steps:
Procedure of Micrografting
Preparation of rootstock
Preparation of scion
Procedure of grafting
Growing of grafted plants in vitro
Transfer to soil

13. Rootstocks used for micrografting are in vitro
germinated seedlings.
When seedling rootstocks are used and all stages
of grafting are conducted in vitro, seeds are
surface sterilized and germinated aseptically in
vessels containing nutrient salts. The seedlings
may be supported on agar medium.
1. Establishment and multiplication of rootstock

14. ‘Carrizo’ seeds in test tubes with
medium for seed germination
Carrizo’ citrange seedlings
growing in vitro in the dark
Carrizo’ citrange seedlings
ready to be used for STG
Diagramatic representation of in vitro seedling
preparation

15. The scion (shoot tip) for shoot-tip grafting can be obtained from various sources of
flushes from selected trees:
directly from field trees that are naturally in flush; however, flushing is season-
associated and suitable plant material from which obtaining the shoot tips is not always
available
•defoliated branches of field trees
•nodal sections cultured in vitro
•budsticks cultured in vitro,
The most commonly used procedure in laboratory is to culture budwoods in vitro at
constant 32°C and exposed 16 h daily to illumination in a culture medium containing
the plant cell culture salt solution of Murashige and Skoog (1962) solidified with 1.2%
Bacto Agar. New flushes are produced in 8-16 days and used for isolation of shoot tips
for STG.
2. Establishment and multiplication of scion

16. Stripped plant producing flushes
Flushes from a budstick.
cultured in vitro
Diagramatic representation of in vitro
and in vivo scion preparation

17. •Flushes cut to about 1 cm long and surface sterilized.
•Shoot tips composed of the apical meristem with three leaf
primordial, measuring 0.1-0.2 mm in length from the cut
surface to the tip of the larger leaf primordia, are excised with a
razor blade sliver attached to a surgical handle.
3. Preparation of rootstock and scion for
micrografting

18. Inverted-T Wedge cut
Diagramatic representation of rootstock and scion preparation
Seedling after growing
in the dark for 14 days (left);
rootstock seedling ready for
performing the graft (right).

19. Meristem, subjacent tissue shoot
tip excised And two primordial
leaves ready to be excised for its
use as scion in STG
Shoot tip excised ready to be grafted
Diagramatic representation of scion preparation

20. The graft type most widely used in STG is the inverted-T incision at the
end of the decapitated epicotyl, although other methods are also used,
such as the wedge cut and the triangular shaped cut.
The shoot tip is placed inside the incision of the rootstock with its cut
surface in contact with the cortex exposed by the horizontal cut of the
incision made at the top of decapitated epicotyl. Following this
procedure in laboratory can obtain about 50% successful grafts.
4. Method of Micrografting

21. Performing the graft

22. Micrografted plants are cultured in a liquid nutrient medium
composed of the plant cell culture salt solution of Murashige and
Skoog.
Keep cultures at around 27°C, exposed daily to 16 hours of light
and 8 h darkness, or natural lighting.
5. Culture of In Vitro Grafted Plants
STG plants growing
in a culture room

23. 6.Transplanting to Soil
Scions of successful grafts should have at least two expanded
leaves before being transplanted to soil.
This stage is usually reached four to six weeks after grafting.
Micrografted plants are transferred to pots containing steam-
sterilized artificial soil mix.
STG plants transferred to pots

24. •The success of this technique is 30-50 per cent and some
time as high as 90 per cent.
•The survival rate of the tips was directly related to their
size.
•Grafting success significantly dependent on the method of
grafting.
Graft Success

25. Advantages
1. The technique is used in citrus to rid plants of viruses and
other systemic pathogens.
2. This technique overcomes problems of producing virus-
free plants from nucellar seedlings or by thermotherapy.
3. Plant obtained by micro-grafting do not have the same
problems as nucellar plants such as reversion to the
juvenile state.

26. 1. Micrografting procedures are difficult and generally results in a low rate of
successful grafts, which makes it an expensive and time-consuming production
technique.
2. It is due to the fact that more technical expertise is required in preparing
successful grafts on small-scale material and handling difficulties associated with
preserving the delicate graft unions.
3. In many experiments, failure rate for micrografts was higher than desired. The
frequency of successful grafts increases with the size of shoot tip, but the
incidence of recovery of healthy plants decreases.
4. In vitro grafts of fruit plants often fail due to incompatibility reaction, poor
contact between stock and scion and phenolic browning of cut surfaces.
Limitations in Micrografting

27. Affecting factors
• Suitability of rootstock: Several factors like type and age
of rootstock, condition of rootstock (etiolated/non
etiolated), and rootstock compatibility with scion
influence the rate of success of grafting and
acclimatization of STG plants.
• Size of meristem.
• Plant growth regulators.
• Environmental conditions.

28. •Browning and tissue blackening
•Sucrose concentration of the medium
•Use of growth regulators
•Pretreatment of shoot apex
•Preventing desiccation of the graft
•Light/dark incubation treatments
•Nature of the supporting medium

29. S.N
o
Fruit crop Scion cultivar Rootstock Successful
micrografts
(%)
Source
1. Pistachio Pistacia vera cv. Mateur Seedling raised rootstocks 94-100 Abousalim and Mantell
(1992)
2. Mulberry Morus alba cv. 707 Seedling raised rootstocks >90 Fengtong et al. (1996)
3. Olive Olea europea cv. Zard In vitro raised seedlings of olive 45-83 Farah et al. (2011)
4. Grape Vitis venifera cv. Early Cardinal 41 B, Salt Creek 71.4 - 80.0 Tangolar et al. (2003)
5. Apple Malus domestica cv. Lal Ambri M-9 rootstock 42.25 Khalid Mushtaq (2009)
6. Hazelnut E-295-S (hazelnut) G-029-N 72.00 Nas and Read (2003)
7. Chestnut 907 (chestnut) 711 80.00 Nas and Read (2003)
8. Walnut Juglans regia cvs. Jinlong No 1,
Xiangling
Seedling raised rootstock 56.70 - 73.30 Wang et al. (2010)
9. Almond Amygdalus Communis cvs.
Ferragnes, Ferraduel
In vitro germinated wild almond
seedlings
90-100 Yildirim et al. (2010)
10. Pear Pyrus communis cv. Le-Cont In vitro shoots of Pyrus
betulaefolia
83.00 Hassanen (2013)
Micrografting success in fruit crops

30. What’s in the future
• Adaptation of shoot tip grafting to more species.
• Micrografting has particular utility in fruit tree
production and protocol should be develop in many
fruit crops.
• Fast and mass propagation of transformed plants.

31. Case studies

32. JagannathUniversity,Jaipur Bishnoi et al., 2017
Conc.
(mg/l)
Per cent grafting
success rate
Boerhaavia diffusa
Control 58.36 ± 2.4365a
5 47.22 ± 1.3900bc
10 33.33 ± 2.4076bc
15 20.83 ± 2.4047d
20 11.11 ± 1.3900e
Clerodendrum aculeatum
Control 58.36 ± 2.4365a
5 43.05 ± 1.3867ab
10 30.55 ± 1.3900bc
15 19.44 ± 1.3867cd
20 13.89 ± 1.3900e
Table 1. Effect of phytoproteins on grafting success in kinnow

33. Rootstock Seedling age (days) Mean
12 14 16 18
Sour orange 16.67 18.33 23.33 28.33 21.67
Carrizo
citrange
38.33 33.33 26.67 16.67 28.75
Rough lemon 26.67 31.67 36.67 18.33 28.33
Cleopatra
mandarin
16.67 23.33 31.67 18.33 22.50
Mean 24.58 26.67 29.58 20.42
CD at 5% Rootstock (R) = 1.64 Seedling age (D) = 1.64 R × D = 3.27
Table 2. Effect of rootstock and age of seedling on STG success (%) in Kinnow
HAU, Hisar Chand et al., 2016

34. Rootstock Days after transfer in soil mixture
15 30 45
Sour orange 58.33 45.83 45.83
Carrizo citrange 83.33 70.83 70.83
Rough lemon 79.17 66.67 66.67
Cleopatra
mandarin
62.50 54.17 54.17
CD at 5% 10.95 8.21 8.21
HAU, Hisar Chand et al., 2016
Table 3. Transplanting success (%) of STG plants on
different rootstocks under greenhouse conditions

35. 35
Age of
seedling
(Days)
Rough lemon Carizo Sour orange Karna Khatta
%
Success
Number
of
days
taken to
bud
sprout
Length of
shoot
%
success
Number
of days
taken
to bud
sprout
Length
of
shoot
%
success
Number
of days
taken
to bud
sprout
Length
of
shoot
%
Success
Number
of
days
taken
to bud
sprout
Length
of
shoot
11
12 42 17.8 1.74
13 36 16.4 1.32 66 18.4 1.88
14 44 16.8 1.70 62 18.4 1.62 54 20.4 1.34 52 20.0 0.98
15 62 17.6 1.88 52 19.4 1.50 50 20.6 1.14 48 20.4 0.86
16 54 19.0 1.46 48 20.4 1.22 48 21.4 0.92 44 20.6 0.72
17 46 19.6 1.06 46 21.0 1.02 44 21.6 0.84 42 22.0 0.66
18 40 20.6 0.76 44 21.4 0.80 42 22.2 0.66
19 36 21.6 0.84 40 22.4 0.78
20 34 22.0 0.74 28 22.6 0.72
SEm+ 1.44 0.21 0.07 1.64 0.21 0.04 1.15 0.19 0.02 0.97 0.11 0.02
CD at 5% 4.13 0.59 0.20 4.70 0.61 0.12 3.29 0.55 0.06 2.78 0.31 0.05
Table 4. Effect of age and type of rootstocks seedling on success of shoot tip
grafting in Blood Red
ARS, Sriganganagar Kanwar et al., 2018

36. CHF, Jhalawar Lahoty et al., 2013
Treatment Success % of shoot tip grafting
Age of rootstock
(days)
Carrizo Rangpur lime Rough lemon Mean
10 0.00 0.00 0.00 0.00
11 26.66 0.00 0.00 8.88
12 43.33 0.00 0.00 14.44
13 53.33 0.00 0.00 17.77
14 33.33 40.00 23.33 32.22
15 23.33 26.66 33.33 27.77
16 10.00 23.33 43.33 25.55
17 0.00 20.00 30.00 16.66
18 0.00 13.33 16.66 10.00
19 0.00 0.00 10.00 3.33
20 0.00 0.00 0.00 0.00
Mean 17.27 11.21 14.24
Rootstock (A) Age of rootstock (B) A × B
CD at 5% 1.91 3.66 6.34
SEm ± 0.67 1.29 2.24
Table 5 : Effect of age and type of rootstocks on success of shoot tip
grafting

37. IBSD, Imphal Sanabam et al ., 2015
Parameter Success rate
Sucrose (%) 1.5 18.45
3 33.62
6 51.62
9 35.62
Rootstocks Kachai lime 28.80
Champra maounthabi 40.72
Solom 32.63
Phouheiree 34.69
Type of incision Inverted T 37.59
Wedge 28.38
Table 6. Influence of different parameters on successful
micrografting of khasi mandarin

38. CCRI, Nagpur Vijayakumari , 2019
S. No. Type of
Budding
Plant
Height
(m)
Stem
Height
(cm)
Stock
Girth
(cm)
Scion
Girth
(cm)
Canopy
Volume
(m3)
No. of
Fruits
1. STG
derived
4.59 38 62.16 58.66 36.49 376.07
2. Convention
al Budded
Plants
4.14 32 57.02 54.83 21.78 155.00
3. C.D. values
at 5%
0.72 NS NS NS 3.95 25.63
Table 7. Comparative field performance of 6 year old STG derived plants Vs.
Conventional budded plants at Sausar (Chhindwara District)

39. Micrografting has great potential for improvement of fruit
plants and has been used for the production of virus and viroid-
free plants in horticultural crops. Besides, it has also been used
in prediction of incompatibility between the grafting partners,
histological studies, virus indexing, production of disease-free
plants particularly resistant to soil borne pathogens, safe
germplasm exchange between countries and multiplication of
difficult to root plants. It is a safe in vitro technique, which can
be utilized for commercial production of virus-free grafted
plants with desired cultivars and suitable rootstock throughout
the year under controlled conditions.
Conclusion