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The present study entitled “Synseed production, in vitro conservation and plant conversion in banana” was carried out at the department of Plant Biotechnology, College of Agriculture,Vellayani during 2014-2016. The objective of the study was to standardize a protocol for synseed production in banana var. Nendran and optimise in vitro conservation procedure for getting maximum conversion to whole plants.
Investigations were carried out in three phases viz. optimisation of encapsulation matrix, regeneration of encapsulated shoot tips (in vitro and ex vitro) and in vitro conservation of encapsulated shoot tips. Shoot tips excised from banana var. Nendran cultures maintained in MS + 2 mg L-1 BA were used as explants.
Four different concentrations of sodium alginate (2.5, 3.0, 3.5 and 4.0 %) and calcium chloride (50, 75, 100 and 200 mM) were tried for encapsulation. Among the 16 treatments, four different combinations (2.5 per cent sodium alginate + 100 mM calcium chloride; 3 per cent sodium alginate + 75 mM calcium chloride; 3.0 per cent sodium alginate + 100 mM calcium chloride and 3.5 per cent sodium alginate + 100 mM calcium chloride) were found to give 100 per cent regeneration, which were on par with the control treatment (non- encapsulated shoot tip). The treatment with 3 per cent sodium alginate and 100 mM calcium chloride was the best, which yielded the maximum number of shoots per explant (3.50), highest number of leaves per shoot (3.74) and the longest shoot (2.41 cm) after 7 weeks of inoculation.
The beads encapsulated with lower concentration of SA (2.5 per cent) germinated by 18th day of inoculation on regeneration medium (MS + 2 mg L-1). Whereas, the beads formed of higher concentration of SA (4 %) took as long as 24 days to germinate. The reduction in ease of bud break due to firmness of the surrounding matrix, especially at higher alginate concentrations might be the reason for delayed germination of beads.
The regeneration of encapsulated shoot tips under ex vitro non- aseptic conditions was tested in four different non- sterile media viz. sand, vermi compost, coirpith compost and potting mixture (sand: vermi compost: coirpith compost at 1:1:1). The beads encapsulated with 4 per cent sodium alginate and 100 mM calcium chloride gave 66.6 per cent regeneration with mean shoot number of 1.0, mean leaf number of 3.5 and longest shoot length of 2.5 cm, in coirpith compost.
The effect of various temperature regimes (4°C, 25°C and -196°C) and duration of storage (0, 2, 4 and 8 weeks) on plantlet regeneration were tested under in vitro conservation studies. Pretreatments like preconditioning, preculture and dehydration, for enabling the explants to tolerate low temperature stresses were standardised.
Preconditioning with 0.5 M sucrose in MS media for 7.0 days was found to be the best. Cent percent regeneration with mean shoots per explant as 4, mean leaves per shoot as 3.3, and maximum shoot length of 2.18 cm were obtained in this treatment.
Shoot tips preconditioned with 0.5 M sucrose for the best duration (7 days) were encapsulated and taken for preculture treatments. Liquid MS medium supplemented with varying concentrations of sucrose (0.5, 0.75 and 1.0 M) were tried for different periods of time (3, 5 and 7 days). Preculture with 0.5 M sucrose for 3 days yielded the best regeneration (100 %), number of shoots per explant (3.21), number of leaves per shoot (3.21) and longest shoot (2.17 cm) .
Preconditioned, encapsulated and precultured beads were dehydrated under sterile air of laminar airflow chamber for 0 to 7 hours. Survival of 61.8 % and regeneration of 51.4 % were obtained in the beads dehydrated for 4 hours (moisture content-18.4 %). The regenerated plants yielded mean shoot number of 1.0, mean leaf number of 1.1 and longest shoot of length 0.76 cm.
The beads could be stored upto 8 weeks with a regeneration of 21.6 per cent at 25°C. At 4°C, storage was possible upto 4 weeks (31.6 % regeneration) beyond which no regeneration was obtained.
Encapsulation dehydration technique was employed for cryopreservation (-196° C). The preconditioned, encapsulated and precultured beads were dehydrated and stored in cryocans containing liquid nitrogen. The cryo plunged beads (1 hour and 2 weeks) were transferred to recovery medium (MS + 2 mg L-1 BA) after thawing (40° C, 60 s). No regeneration was obtained. After storage, blackening of encapsulated tissues was observed and no regeneration was obtained.
Insufficient pretreatments of explants prior to cryopreservation might be a reason for failure of regeneration of cryo plunged beads. According to Panis (2009), partially or completely black shoot tips of Musa sp. following cryopreservation indicate the occurrence of enzymatic reaction. He also opined that AAB plantains and diploids generally respond poorly to freezing.
The encapsulated shoot tips could be regenerated after 8 weeks of storage at 25° C. Encapsulation technique can thus be successfully used as a method for germplasm conservation and exchange. Conservation and propagation of vegetatively propagated plant species and plant lines that are difficult to propagate through conventional methods are made easier through synseed technology.
The possible future lines of this work are: modification of pretreatments for getting survival & regeneration after cryopreservation, studies on the effect of size of explants used on regeneration and plantlet conversion, investigating the effect of preconditioned, encapsulated and precultured beads in imparting resistance against abiotic stress, studies on enhanced longevity of beads under high temperature compared to low temperature.