In Vitro Multiplication from Nodal Explant of Roseapple and Genetic Purity Study of Conserved Plantlets Using Molecular Marker

In vitro multiplication, an exceptional replacement for conventional propagation practices as it produces maximum plantlets in minimum space and time. In vitro multiplication in rose apple was attempted through direct organogenesis of nodal explant. During shoot multiplication the highest numbers of shoots with the maximum number of leaves were achieved in full MS as well as in half MS with 4 mg/l BAP supplementation but half MS were recommended for better survival of plants as it lowered the polyphenol exudates interfering plant survival. In vitro root induction was achieved within two weeks of shootlet cultured on half MS with IBA at 6 mg/l concentration. Shoot multiplication from nodal explant produced on an average 30 plantlets from a single seed within 4-5 months. In vitro produced as well as conserved plantlets showed no genetic differences when it is compared to in vivo regenerated plants by using RAPD markers.

Roseapple (Syzygium jambos L.), a fruit tree, known as malabar plum, gulab jamun etc., a dicotyledonous plant, belonging to family Myrtaceae, native to south-east Asia, is widely grown in tropical and subtropical region. Fruits with a characteristic odour are consumed fresh as well as processed. Roseapple has many medicinal properties. It reduces fever, controls diarrhoea and diabetes. It is usually propagated by seeds. Being polyembryony, a single seed may produce as many as four seedlings 1 . For rapid and true to type multiplication at any time and place in large number, in vitro organ culture is a successful practical approach. But in vitro studies in roseapple are very limited. Germination of immature adventitious embryo on MS medium with low concentration of BAP or without any PGR was reported by Litz 2 . In the recent past Prashanta et al. did successful callus culture and plantlet regeneration 3 . The objectives of the present study include in vitro propagation and multiplication from nodal explants. Simultaneously genetic purity study using molecular marker has been attempted in in vitro cultured short term or medium term conserved plantlets.

MATERIALS AND METHODS
Ripened fruits were collected from different growing areas of South 24 Parganas, West Bengal, India. They were thoroughly washed, cleaned, dried and imbibed in water for overnight and surface sterilized with 1% (v/v) savlon + 1% (v/v) Tween 20 for 30 minutes, washed 4 times in distilled water, also treated with 1% (w/v) Bavistin for 10 minutes, washed for 4 times in distilled water. They were then treated with 0.1% (w/v) Mercuric Chloride Print ISSN : 2347-9655 Online ISSN : 2454-9541 (HgCl 2 ) for 10 minutes and washed for 4 times with distilled water.
Surface sterilized seeds were cultured in 500 ml culture vessels containing 40 ml of MS semisolid media with 30g/l sucrose, 100 mg/l myoinositol without any PGR. The collected data were analysed using one way analysis of variance (ANOVA). ANOVA of % survival was done after square root transformation of % data. Treatment means were compared based on Least Significant Difference (LSD) through AGRES (version 7.0) software package.
Root induction was recorded within 2-3 weeks of culturing on half MS supplemented with either 6 or 8 mg/l IBA. It was found that both 6 mg/l and 8 mg/l IBA produced 12-15 robust roots of about 1 cm length within 7-8 weeks of culture (Fig.  1D). No significant differences between these two concentrations were found. It can be suggested that 6 mg/l IBA is good enough to induce root in vitro. This confirms earlier observation of Prashanta et al. 3 .

Genetic fidelity test
RAPD primers are easy to handle molecular markers which do not require any knowledge of DNA sequence of targeted genome. The primer will bind randomly in the sequence. However it relies on a large, intact DNA samples and its resolving power is also much lower than target specific DNA comparison methods. Because of its simplicity and random DNA binding capacity Shakya (Fig. 1E). Thus, it can be concluded that derived planlets through direct organogenesis in vitro and conserved plantlets in vitro, atleast for a period of 1 year do not produce any genetic changes. However with the inherent limitation of RAPD as molecular marker present observations need to be considered.

CONCLUSION
We have seen that most shoot growth characters ran parallel in both half and full strength MS. But in case of shootlet survival, half MS showed far better result than full MS. Such observation is not unexpected. The reduced MS strength to half inhibits polyphenol exudates which helps promote survivality. This is also observed by Qureshi et al. 4 . Apart from this, bud break also took place earlier in half MS, which shortens the time period for multiplication.
BAP concentration is proved to be another key player in plantlet regeneration and multiplication. BAP concentration at 4 mg/l showed the best result in terms of shootlet growth characters, while comparing with others. Thus, considering both shootlet growth and survival, half MS with 4 mg/l BAP appeared to be desirable media formulation. Interestingly Prashanta et al. also observed 4 mg/l BAP as favourable concentration for shootlet growth of S. jambos, but his study was confined to full MS 3 .
Different events over different time schedule during the course of nodal explant multiplication in vitro (bud break-shootlet-rooting) have been summerized (Table 4). It becomes evident that it took around 16-20 weeks i.e. around four and half months to raise complete plantlets from the nodal explants. It is to be noted that as many as 7-8 shootlets were induced in vitro in MS without PGR from a single seed. During the course of the study, altogether 4 nodal explants were derived from a single shoot. Based on these facts, it is possible to propagate as many as 28-32 plants from a single seed through nodal explants multiplication in vitro. This in vitro multiplied shootlets did not show any genetic variability as confirmed by the genetic fidelity test. So, from these observations, it can be concluded, roseapple can be multiplied within a short time span and conserved in vitro (atleast one year) for continuous supply to the farmers without hampering its genetic makeup.