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Micropropagation of Artocarpus lakoocha Roxb (Monkey jack) through Shoot Bud Culture  

Geetika Pant1 , Umesh Kabade2
1. Department of Biotechnology, Indian Academy Group of Institutions, Bangalore, India
2. Plant Tissue Culture, Arya Agribiotech and Research Centre, Santokpura, Anand, Gujarat, India
Author    Correspondence author
Tree Genetics and Molecular Breeding, 2014, Vol. 4, No. 1   doi: 10.5376/tgmb.2014.04.0001
Received: 06 Mar., 2014    Accepted: 11 Mar., 2014    Published: 17 Mar., 2014
© 2014 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Pant et al., 2014, Micropropagation of Artocarpus lakoocha Roxb (Monkey jack) through Shoot Bud Culture, Tree Genetics and Molecular Breeding, Vol.4, No.1-6 (doi: 10.5376/tgmb.2014.04.0001)

Abstract

The Lakoocha (Artocarpus lakoocha Roxb.), popularly known as “monkey jack”, is called as ‘barhal’ in India, belongs to family Moraceae and comprises of many species of woody trees. The present study was aimed to develop a micropropagation protocol for Lakoocha towards commercial mass production of planting material which permits rapid and easy multiplication rates, outwits genotype barriers, offers opportunities for continuous availability of genuine planting material with economic viability. We report here a prototype system of micropropagated plantlets of Lakoocha through shoot bud culture within 6 months catering to those parameters of commercial mass multiplication. Nodal explant with an axillary bud used as explant; showed in vitro bud induction within 7.33days in MS+BAP 13.33+IAA 2.28+GA3 0.57 µM. The maximum shoot multiplication (6.66 shoots/explant) was observed in MS medium supplemented with BAP 13.33 +IAA 1.14 µM. The microshoots were maintained in MS basal medium (BAP 0.89 µM) supplemented with Casein Hydrolysate 100 mg/l and then subsequently rooted in ½ MS medium supplemented with IBA 9.85+BAP0.89 µM+Activated charcoal 500 mg/l which resulted in 88.16% rooting. The in vitro rooted plants were primary hardened on autoclaved Sand : Soil : Peat moss (1:1:2) drenched with  ½ strength MS nutrients in plastic cups covered with transparent polyethylene bags to prevent excess water loss for 3 weeks in growth room at 25±20C and then transferred to shade net house (50% shade 70%±5% RH) for secondary hardening. All plants showed 88.33% survival during acclimatization. The plants that were field established showed > 85% survivability, which is under further evaluation for fruiting and quality characters.

Keywords
Prototype; Micropropagation; Artocarpus lakoocha; Monkey Jac

The lakoocha (Artocarpus lakoocha Roxb.), most popularly known as “monkey jack” or ‘barhal’ and pachoo phanas in local language (bangalore) in India, belonging to family moraceae, originated from South East Asia and is widely distributed in the humid sub-himalayan regions of India. Itis regarded as underutilized fruit, although “lakoocha” is grown mainly in Uttar Pradesh, Bengal, Khasi Hills and Western Ghats, because of lack of commercial orchards, lesser consumer preference and poor marketability. Recent commercial interest in several tropical and subtropical underutilized fruits has resulted in an increased cultivation area in Asia and other regions of the developing world due to high level of vitamins and minerals has been recorded in a number of underutilized fruits. The export of fruits from Asia alone has been increasing by a little over 10% annually (Singh, 1993). Most commonly, leaves of these trees serves as green fodder for cattle due to their rich source of crude protein, fibers and mineral (Joshee et al.,2002). These trees grow upto 6 to 12 m tall with large, leathery and deciduous leaves but with fruits of no edible value. Yet the choice of the fruit in Ayurveda and Siddha medicines are due to their multivarious active principles. The medicinal principles that have been well documented in A. lakoocha includes tannins (Doss et al., 2009), flavonoids (Mandalari et al., 2007; Maneechai et al., 2012), saponins (Avato et al., 2006), terpenoids (Funatogawa et al., 2004) and alkaloids (Navarro et al., 1999). Fruit pulp is used as liver tonic and bark extract is used in external ailments like heals boils, cracked skin and pimples (Shailendraet al., 2010). The stem is vermifuge and the stem bark is useful in curing tapeworm infection (Sambhandharaksa et al., 1962, Charoenlarp et al., 1981). A. lakoocha extract is also reported to have cosmetic ingredients (Tengamnuay et al., 2006).

With all these potential uses in pharmaceutical industry, there is a need for commercial orcharding and area expansion for which conventional methods of sexual and vegetative propagation have proved to be futile because of the inherent properties of poor seed viability and low germination percentage and poor response to vegetative means of cutting or grafting techniques (Napier and Robbins, 1989). This prompts for alternative approaches of developing propagules of A. lakoocha in large scale and micropropagation has been a promising tool that has been reported to be fruitful in developing successful plantlets.Where in the conventional propagation methods had been less successful or resulted in complete failure. There are few reports on micropropagation of A. lakoocha using in vitro raised seedlings (Joshee et al., 2002; Rahman and Amin, 1995) and some reports on jackfruits (Artocarpus heterophyllus Lam.) by Abd El-Zaher (2008), Khan et al. (2010), Ashrafuzzaman et al. (2012). We report a rapid and efficient micropro- pagation protocol using nodal explants from 8 years old tree, for producing higher number of plantlets. This prototype system will facilitate commercial production of micropropagated plantletsof A. lakoocha as a source of providing genuine planting material.
1 Result and Analysis
1.1 Shoot bud induction and multiplication
Explants cultured in MS+BAP 13.33+IAA 2.28+GA3 0.577 µM recorded early bud induction in about 7.33 days which showed longer axillary shoot (4.567 cm/ explant) and heighest number of leaves (6.667 leaves/explant) (Table 1) than other treatments. However, MS+BAP 13.33+IAA 1.14 µM containing multiplication medium produced 6.667 microshoots/ explant which also showed maximum number of leaves (8.667 leaves/explant) and microshoot length (3.967 cm/explant) (Table 2).


Table 1 Effect of different concentration of plant hormones on in vitro shoot bud induction


Table 2 Effect ofdifferent concentration of cytokinin and auxin ratio on shoot multiplication

1.2 In vitro
rooting in regenerated shoots
In vitro rooting was dependent on the strength of the medium and the auxin concentration as evident from Table 3. Among the various strength of media evaluated, IBA, IAA and BAP levels; ½ MS+IBA 8.88+IAA 1.14+BAP 0.89 µM+Activated charcoal 500 mg/L produced 88.16% rooting with heighest number of roots (4.33 roots/explant) over other treat- ments. In MS+IBA 13.33+IAA 1.14+BAP 0.89 µM, regenerated shoots produced longer roots (5.26 cm).


Table 3 Effect of different concentration of auxins and cytokinins on in vitro rooting

1.3 Hardening of in vitro plantlets
Acclimatization of the rooted plantlets was tested in various carrier substrates (Figure 1). A combination of Soil+Sand+Peat moss (1:1:2) supplemented with ½ MS nutrient solution was identified to be ideal substrate for acclimatization, as this treatment resulted in maximum plant survival rate (88.33%), longer plants (6.56 cm) with healthy and green canopy of leaves (8.33 leaves/plant). Although, longer roots (5.96 cm) were found in coconut husk carrier this treatment did not show efficient survival rates. Totally 46 plantlets survived in the greenhouse after one year.


Figure 1 Effect of carrier substrates on acclimatization of micropropagated plantlets

2 Discussion
2.1 Shoot bud induction and multiplication
Micropropagation of mature trees using vegetative parts as explants has been a difficult task due to various factors, like juvenility vs. maturity, inherent slow growing habit, exogenous and endogenous infection, presence of phenolic compounds, long complex life cycles, great genetic variations, etc. (Bonga and Durzan, 1986; Bajaj, 199; Ali et al., 2003; Liu and Yang, 2011). In vitro propagation of plant species is influenced by factors such as genotype, age and source of initial tissue/organ which in turn are related to their endogenous hormonal status (George, 1993). Most of the hardwood species produce phenolic compounds after wounding (George and Sherrington, 1984) which is also encountered in A. lakoocha. The phenolic substances on oxidation get converted into quinines which cause tissue blackening and inhibit new in vitro morphogenetic responses in plants. Phenolics could be minimized by keeping explants right from the time of collection to inoculation in antioxidant solution comprising of ascorbic acid, citric acid and PVP (Elmore et al., 1990; Liu and Yang, 2011). Polyvinylpyrollidine (PVP) is a polyamide which absorbs phenols through hydrogen bonding and thus prevents their oxidation. It is the mostly commonly used antioxidant in micro- propagation of hard wood trees like neem (Gautam et al. 1993); ascorbic acid was also reported to be potent antioxidant by Pati et al. (2008) and Abd El-Zaher (2008) during micropropagation process. In the present study, theexplants of A. lakoocha were given a pretreatment with PVP (25 µM) for one hour which was found to be effective in controlling oxidative browning.
Explants play major role in developing a prototype system for micropropagation of perennial hard wood trees. Different types of explants were tried by several research groups. Earlier, nodal stem segments were reported by and Pati et al. (2008) and Raj and Basavaraju (2012) in micropropagation of A. mormelos, an underutiliszed fruit crop;which proved to be successful because of the presence of protected axillary buds unaffected by toxicity due to surface sterilants and regeneration of true to type plantlets.
Optimization of explants was important for successful micropropagtion technique as this forms the prime factor for development of an efficient prototype system. Therefore, preliminary studies on establish- ment of aseptic cultures with ideal explants were carried out in A. lakoocha. Explants of mature lakoocha tree exhibit high level of exogenous and endogenous contamination. Surface sterilization with 0.1% mercuric chloride (HgCl2) for 8 min was found to be most effective for reducing contamination or infection levels in nodal stem segments in lakoocha. These results were on par with the earlier reports on surface sterilization using 0.1% mercuric chloride in Crataeva nurvala (Sharma and Pandey, 1996).
Media is another critical factor that positively or negatively influences the in vitro regeneration. In the present study, MS+BAP 13.33+IAA 2.28+GA3 0.57 µM was found to be statistically significant in terms of bud induction, longer axillary shoots and heighest number of leaves/explant of in vitro established culture. Hamid et al. (2007) reported that jackfruit explants cultured on MS+BAP 13.33+NAA 0.53µM produced the highest number of shoots, leaves/explant and shoot length. However, a combination of BAP with IAA (MS+BAP 13.33+IAA 1.14 µM) augmented multiplication of shoots. The synergetic effect of auxin along with cytokinins on shoot multiplication and shoot bud induction has been reported by several workers (Mannan et al, 2006; Abd El-Zaher 2008).
2.2 In vitro rooting in regenerated shoots
Finally, the microshoots were tested for rooting efficiency in medium with various auxin levels. Microshoots incubated on ½ MS IBA 9.85+IAA 1.14+ BAP 0.89 µM+Activated charcoal 500 mg/L signify- cantly enhanced the root induction (88.16%) and number (4.33). Roy et al. (1996) obtained successful rooting of in vitro shoots of jackfruit on ½ MS+ IBA 4.9 + NAA 5.37 µM. Most recently, Khan et al. (2010) have reported higher rooting rate (80%) and (6.9) numbers of roots per plant were obtained with IBA 14.77 µM in jack fruit.
2.3 Hardening of in vitro plantlets  
Plantlets grown in vitro have been continuously exposed to controlled conditions for rapid plant growth and sudden transfer is impossible directly to pot which causes maximum mortality of micropropagated plants. A gradual acclimatization to natural environment is needed to increase survival rates and hence, an intermediary greenhouse hardening is done which depends mainly on the carrier substrates (Hazarika, 2003). Sand+ Soil+ Peat moss as carrier substrate with ½ strength MS nutrients has marked effect on the plant height, number of leaves/explant and overall survival of plants in lakoocha which was coinciding with the results in jackfruit (Roy et al., 1993; 1996). Acclimatization procedures have been attempted to increase ex vitro survival of plantlets upon transplanting (Roy et al., 1990; 1996; Amin and Jaiswal, 1993; Noweret et al., 2005; Pati et al., 2013). The in vitro raised plants showed profused growth when transplanted in soil.
The protocol establishes here is highly reproducible for the production of plantlets which is not possible with conventional methods. Plant growth regulators and the physiological activity of the explants are very important for successful regeneration. The plants that were under further evaluation for fruiting and quality characters. The plant regeneration protocol established in this investigation may facilitate future research in genetic transformation in lakoocha and related genus.
3 Materials andMethods
3.1 Establishment of culture
New shoots of 15~20 cm sizes from 8 year old tree of Artocarpus lakoocha Roxb were defoliated and collected in an aqueous solution of ascorbic acid  (5.78 µM) and brought to the laboratory for further processing. The nodal explants were washed thoroughly under running tap water for 1h and treated with Reidomyl 0.1%+Timentin 1.05 mM in 100 ml of distilled water and 2 drops of Tween-20 for one hour to avoid bacterial and fungal pathogens. The segments were washed 3~4 times using sterile distilled water. A pre-treatment with chilled antioxidant solution comprising ascorbic acid (5.68 mM) and Polyvinylpyrollidine (25 µM) for 1 h was also followed in order to minimize in vitro oxidative browning due to phenolic compounds. Then the explants were rinsed with sterile double distilled water and taken to laminar hood for surface sterilization. Shoot buds were dipped in 70% ethanol for 30 seconds followed by air dried. The explants were treated with 0.1% HgCl2 for 8 minutes and then washed (3~4 times) with sterile distilled water and blotted dry on pre sterilized filter paper sheets.
Surface sterilized explants were placed vertically on MS (Murashige and Skoog, 1962) medium supple- mented with BAP (0, 2.22 µM, 4.44 µM, 8.88 µM and 13.33 µM), GA3 (0.57 µM), ADS (2.71 µM) and IAA (1.14 µM and 2.28 µM) for shoot bud induction and multiplication. All the media were added with 0.7% (w/v) agar and 3% (w/v) sucrose. The medium was changed 2~3 times over the first 10~14 days of the study to control phenolics exudates to establish cultures. Observations were recorded on morph- genetic characters such as days taken for bud break, number of leaf per explant, length of axillary shoots (cm) and average number of microshoots per explant after 30 days of culture of the explants.
3.2 In vitro rooting in regenerated shoots
Microshoots obtained in vitro were subjected to auxins and cytokine such as IBA (0, 9.85 µM, 14.77 µM and 19.70 µM), IAA (1.14 µM) and BAP (0.89 µM) in half and full strength of MS basal medium supplemented with sucrose 3%, activated charcoal 500 mg/l and agar 0.7%. Data were recorded on per cent rooting, number of roots, and length of roots. All cultures were incubated at (25±2)0C and were exposed to a photoperiod of 16/8 hours light and dark cycling in the culture room by cool white fluorescent tubes (80 mmol m2s-1) with 70%±5% relative humidity. 
3.3 Hardening of in vitro plantlets
Eight week old rooted shoots, were shifted to six inch plastic cups for acclimatized filled with autoclaved Sand:Soil:Peat moss (1:1:2) and drenched with ½ strength MS nutrients. The plastic cups was covered with transparent polyethylene bags to prevent excess water loss for 3 weeks in growth room at (25±2)0C and then transferred to shade net house (100 mmol m2s-1; 50% shade 70%±5% RH) for secondary hardening. Survival percent were determined before field plantation. When 6~8 leaves emerged in the plants, they were shifted to field.
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