Abstract
High level of salinity in coastal areas hinders fodder production for livestock in Bangladesh.
Development of salt tolerant variety of high yielding fodder is a crying need of time for the
sake of livestock development in the areas possessing salt in soil and water. To develop saline
stress tolerant fodder variety, genetic manipulation through gamma irradiation (Gys) and salt
tolerant gene transformation in Napier (Pennisetum purpureum) fodder was conducted. Sixty
double node cuttings of 2 Napier cultivars (BN-3 and BN-4) developed at Bangladesh Livestock
Research Institute were treated with 10-100 Gys at 10 doses intervals. On the basis of plant
growth and profuse tillering, best clumps of Line-1 and Line-2 (10 Gys), Line-3 and Line-4 (20
Gys), Line-5 and Line-6 (30 Gys), Line-7 (40 Gys), Line-8 (50 Gys) and Line-9 (60 Gys) were
selected in 1st mutagenic generation (M1). Based on superior performance, 7 lines (Line-1 to
Line-7) were chosen for re-plantation to produce M2. Genetic diversity of 9 mutant lines in M1
along with a control line (without radiation) was determined by using 22 RAPD markers.
Callus induction, plantlets and shoot regeneration were derived from leaf roll segments in MS
media supplemented with 2,4-D, NAA and BAP and also coconut water. Salt tolerant AtNHX3
gene was transformed through Agrobacterium in BN-4. Along with a control line, 5 mutant Lines
(2, 3, 4, 5 and 6) from BN-3 and 4 mutant Lines (3, 4, 5 and 6) from BN-4 were selected based on
performance and genetic diversity. In addition, a transgenic line from BN-4 was selected for
testing salt tolerance in hydroponic condition. Along with a control line, 3 mutant lines from
BN-3; Line-2 (exposed to 10 Gys), Line-4 (20 Gys), and Line-5 (30 Gys) and 3 mutant lines from
BN-4; Line-3 (exposed to 20 Gys) and Line-5 and Line-6 (both exposed to 30 Gys) were selected
on the basis of salt tolerance level at 12 dSm-1 in hydroponic condition. The results revealed that
higher Gys delayed shoot initiation as well as decreased number of shoots. The LD50 in BN-3
and BN-4 were 28.2 and 27.5 Gys, respectively. The production and nutrient contents showed
significant (p<0.05; p<0.01) variations among lines in M1 and M2 generations for both cultivars.
In the genetic diversity study, overall polymorphism (%), average gene frequency, expected
gene diversity and genetic distance in BN-3 were 30.67, 1.30, 0.18 and 0.017-0.342, respectively,
while in BN-4 the same were 25.25, 1.25, 0.16 and 0.018-0.266. Cluster analysis clearly showed
formation of sub-clusters under principal cluster and sub-clusters further grouped into subclusters among lines for both cultivars, which revealed genetic variations due to exposing Gys.
In tissue culture experiment, hormone concentration in MS medium had significant (p<0.05;
p<0.01) effect on rate and time of callus induction, plantlet regeneration, multiple shoot
formation and time of plantlet regeneration. The highest callus induction in BN-3 and BN-4
(84.71 and 82.59%) and time required for callus induction (23.14 and 21.66 days) were obtained
in 2,4-D at 2.0 ppm. On the other hand, highest plantlet regeneration for BN-3 and BN-4 (55.33
and 56.66%), multiple shoot formation (74.13 and 78.26%) and time of plantlet regeneration
(30.0 and 30.66 days) were obtained in NAA (1.0 ppm) +BAP (0.5 ppm). The overall
Agrobacterium transformation efficiency in BN-4 was better (5.28%) at 2,4-D (0.0 ppm)+NAA
(1.0ppm)+BAP (0.5ppm) hormone combination added with 100 ppm Acetosyringone at the calli
age of 30 days and duration of infection and co-cultivation times of 20 minutes and 3 days,
respectively. In hydroponic condition, production and nutrient contents varied significantly
(p<0.01) among lines in both cultivars, where less Na+ (mg g¹) was accumulated in Line-5
(0.324 in BN-3) and Line-3 (0.349 in BN-4) at 12 dSm-1. In field trial at coastal region, there were
significant (p<0.01, p<0.05) variations of production and CP contents due to effect of lines and
seasons; the highest plant survivability (%), biomass yield (t/ha/cut), tiller (no/clump), plant
height (cm) and CP (%) in dry season were obtained from Line-5 of BN-3 (74.5, 21.13, 32.0,
132.58 and 12.10, respectively) and Line-3 of BN-4 (72.50, 21.94, 24.67, 140.21 and 11.33,
respectively). In the summing up, all mutant lines developed from both cultivars performed
better than control line in terms of plant morphology, yield and CP content. However, mutant
lines exposed with 20-30 Gys had shown to be superior survivability and production
performance in dry season when salinity level increases.