Micro-Propagation and Bio-priming in Pomegranate - Imperative for Quality Planting Material

Pomegranate is one of the ancient fruit crops domesticated by the mankind and is considered as a highly remunerative fruit crop under semiarid tropics. The versatile adaptability and hardy nature of the crop support fruit yield by overcoming climatic vagaries and edaphic challenges of the semi-arid regions [1]. The unmatched nutraceutical and therapeutic properties of this super fruit has attracted consumers across the globe creating a huge demand for its fruits, value added products and dietary supplements. Globally pomegranate is cultivated in about 0.5 million ha area with an annual production of approximately 6.0 million tonnes. In India, pomegranate is cultivated in about 0.234 million hectares land with an annual production of 2.85 million MT. There is significant growth in acreage and production of pomegranate globally in the recent past and India has almost registered an impressive 3 folds increase in acreage and about four folds increase in production of pomegranate during last two decades. Furthermore, the export is estimated to be 0.67 lakh MT. The average annual increase in pomegranate acreage during last 10 years in India is about 19,300 hectares, and the average annual quantity of healthy saplings required to plant this area is about 15.0 million [2]. Though, conventional methods of propagation are commercially used for propagation of pomegranate saplings, but these methods do not ensure elite and healthy/ disease free/ blight free saplings production. The bacterial blight, wilt and other sapling or potting mixture transmitted diseases are major threats for the sustainable pomegranate production and use of in-vitro conditions to extend pomegranate clonal propagation has allowed for rapid mass multiplication of clonal propagation of superior plant material which are free from pathogens causing blight and wilt diseases in pomegranate. The ultimate objective is to extend pomegranate agriculture area all over the world, which demands large scale and disease-free clonal propagation of superior genotypes using micropropagation.

Micro-propagation is a plant tissue culture technique used to produce plantlets in which aseptic tiny portions of tissues and organs are cultured in vessels with predetermined culture media and under controlled environmental conditions to develop plantlets.

There are five distinct stages in micro-propagation:
1. Stage 0: It involves the selection and maintenance of elite and healthy mother plant of a commercial variety and explant selection and pretreatments
2. Stage I: In vitro culture establishment
3. Stage II: In vitro shoot proliferation
4. Stage III: In vitro rooting
5. Stage IV: hardening or acclimatization

The source of explants, explant type and pretreatments given to the explants are considered as crucial variables for in vitro culture establishment and success of micro-propagation protocol of pomegranate. In terms of regeneracy, not all explants are supposed to be equal. Commercial micropropagation includes meristems, shoot tips, and nodal buds [3-9]. However, in vitro regeneration using other explants like leaf segments and cotyledons [10-13], anthers [14] or through embryogenesis from various seedling explants, petals, and immature zygotic embryos [10] were also reported. The stage 0 also entails identifying healthy mother plants and treating them in such a method that they produce more responsive explants suited for establishment in contamination-free cultures. For the culture establishment appropriate explants are selected, sterilised, and transferred to nutrient media. Soaking of nodal segments in fungicides @0.1-0.2% (Dithane M- 45 and/or Carbendazim) solution for about 30-45 min followed by treatment with antibiotics (streptomycin solution @0.05-0.1%) for 10-20 min. and surface sterilization for 5-10 min. take care of most of the infections [6,7]. Most widely used surface sterilants in pomegranate are HgCl2 and NaOCl. The explants are washed thoroughly under running tap water before the surface sterilization process [15].

In vitro propagation of pomegranate is mostly carried out on modified Murashige Skoog (MS) and Woody Plant Medium (WPM) [2,9]. The ratio of cytokinin to auxin determines shoot proliferation in pomegranate micropropagation. Among cytokinins, 6-Benzylaminopurine (6-BAP) and Kinetin and cytokinin supplements like Adenine Sulphate are mostly used for culture establishment and shoot proliferation and among auxins α-naptheleneacetic acid is commonly used [14,16,2]. The proliferation rate (10-15 shoots/explant) of the Bhagwa nodal segment at the establishment stage was highest on MS media with 1.8mg/l BAP, 0.9mg/l NAA, 1mg/l silver nitrate (AgNO3), and 30mg/l adenine sulphate [6]. Two types of basal medium – MS and WPM had been used for culture establishment of nodal segments and shoot tips of pomegranate cvs. “Malas Saveh” and “Yousef Khani” [17].

Establishment of woody plants like pomegranate is severely affected by browning of medium due to leaching of secondary metabolites like phenols. Culture of juvenile explants, or new growth flushes during the active growth period, culture in darkness, transfer to inoculums in fresh medium at short intervals (rapid sub-culturing), culture in liquid medium with antioxidants in the culture medium, or soaking explants in water or antioxidantcontaining solutions prior to inoculation, supplementing the medium with adsorbing agents like Activated Charcoal, PVP (polyvinyl pyrollidone), etc are some of the different approaches for controlling the browning of growth medium. For controlling the media browning, shoot tips and nodal segments of pomegranate cv. ‘Ganesh’ in vitro are obtained by rapid subculturing [12]. Rapid subculturing on first and third day after inoculation was suggested for in vitro propagation of pomegranate cv. ‘Mridula’ with the use of shoot tips and nodal segments of mature trees as explants [3]. The phenol exudation could be reduced if the cut edges of nodal segments are sealed with sterile wax [8]. In vitro rooting was best obtained on half strength MS medium containing 500mg/l of activated charcoal [18].

The extent and rate of multiplication is influenced by the duration and concentration of auxins and cytokinins in the multiplication medium. In a variety of plants, the cytokinin 6-benzylaminopurine (BAP) is the most often utilized growth regulator for shoot regeneration. Using cotyledonary nodes produced from axenic seedlings, a full procedure for in vitro regeneration of pomegranate (Punica granatum L.) was studied [14]. On a medium containing 9.0μM BA, the largest number of shoots (9.8 shoots/explant) was generated [14]. The treatment MS + BAP 2.0mg/l had the highest number of shoots per explant (1.73), whereas the medium containing MS + BAP 2.5mg/l had the highest shoot length and number of leaves [15]. MS medium supplemented with 2.0mg/l 6-benzylaminopurine (BAP) and 1.5mg/l naphthalene acetic acid (NAA) resulted in the maximum frequency of shoot regeneration (67.89%) along with the average shoot length of 3.62cm and the number of shoots generated (5.38) per explant [19]. Under both liquid and solidified Murashige and Skoog (1962) media, the optimum hormonal treatments for direct adventitious shoot regeneration in cotyledonary explants [20]. The micro-shoots of cvs. “Malas Yazdi” and “Shirine Shahvar” were multiplied in vitro, and the highest shoot height was reported on MS medium containing 2mg/l BA [17].

Various level of light intensity with a constant temperature 26±2° C and 16/8h light/dark period in the incubation room were studied and at 3000 lux intensity, the largest number of shoots per explant and the longest length of shoots were reported [8].

During stage III, the proliferating shoots are placed to a rooting medium. It is the final stage in vitro before the plantlets are transferred to ex vitro environments. The goal of this phase is to get fully grown plantlets to start growing. Auxins are primarily concerned with in vitro root induction. Among all auxin varieties, the auxin IBA was shown to be the most effective. IAA, on the other hand, although being natural, was the least effective since it was damaged by light. The salt content in rooting media is often low. Half-strength WPM medium supplemented with 0.54μ M NAA was the most effective for rooting of microshoots in cvs. “Malas Yazdi” and “Shirine Shahvar” [21]. Half strength MS medium supplemented with 0.1mg/l IBA was the most successful in inducing rooting of microshoots. In vitro produced shoots in half strength MS media with 1.0mg/l IBA promoted strong roots (80%) in 8-10 days [22]. Good in vitro rooting was observed on half strength MS medium supplemented with 0.5g/l of IBA [12]. According to Singh and Khawale, the MS medium containing 2.0mg/l IBA, 200mg/l AC, and 40g/1 sucrose increased root quality [23]. In comparison to control, a considerable increase in average root number per shoot (7.4) was observed after a brief dip of in vitro produced shoots in 1000mg/ lit IBA [24]. Half strength MS medium with 0.1mg/l NAA and 0.02% activated charcoal was found to be the most efficient for rooting shoots, resulting in an 80.12 % root regeneration frequency [19].

Hardening is the process of acclimating in vitro grown plantlets to regular outdoor conditions by gradually exposing them to diffused light in humid chambers. Hardening in vitro grown plantlets is critical for their long-term survival and establishment. Ex vitro survival of cv. “Ganesh” plantlets was reported to be 68 % when transplanted to vermicompost [12]. The plantlets in cv. “G- 137” had the highest survival rate (89%) and took the shortest time to field transfer (35 days) [8]. The method of using a glass jar with a polypropylene cap packed with wet peat: Soilrite® (1:1) was shown to be the most effective, resulting in the highest plantlet survival rate of 86.5 % of cv. G-137 in vitro raised plants [23]. The rooted micro-cuttings registered 80% survival when shifted to a mixture vermiculite (60%), perlite (30%), and cocopeat (10%) v/v [25]. Acclimatization of regenerated plantlets in plastic cups containing autoclaved peat moss resulted into 85 % survival rate [19]. Pots containing sterilized cocopeat-perlite mixture and covered with polythene bags was successfully used for hardening for in vitro rooted plantlets, plantlets were kept at 25± 1ºC in artificial light (50μmol m-2s-1) for 3-4 weeks for proper hardening (10). Commercially, primary hardening of in vitro raised rooted pomegranate plantlets in India is carried out in nursery trays or net pots and hardened under low tunnel polyhouses with very high relative humidity. Bio-priming is utilization of plant beneficial microbes and/or their formulations and inoculating them in rhizosphere and/or phyllosphere of in vitro raised plants or saplings at in vitro or ex vitro or nursery stage to improve field survival and performance inoculated saplings [26,9]. The bioprimed/ biohardened plantlets exhibit higher survival, more root and shoot biomass production, enhanced photosynthesis, and better nutrient uptake [27-33,9].

To date, significant progress has been made in the creation of pomegranate in vitro plant regeneration systems that are fast, repeatable, and dependable. To meet out the demands of rapid expansion in pomegranate cultivation a commensuration production of Quality Planting Material (QPM) is compulsory for sustainable production.

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