Standardisation of tissue/meristem culture techniques in important horticultural crops
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Date
1985
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Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara
Abstract
Attempts were made in the Plant Tissue Culture Laboratory of the College of
Horticulture, during 1981-85 to standardize tissue culture techniques for the
propagation of some of the important horticultural crops of Kerala.
Explants from shoot apices of fresh stem sprouts of five year old jack (Artocarpus
heterophyllus Lam.) trees registered a multiplication rate of 4.5 x when cultured for
five weeks and produced 70% rooting (in 13.43 days) with 5.43 roots per shoot. The
MS medium supplemented with GA 1.0ppm and activated charcoal 1.0% was
identified as suitable for culture establishment, supporting the survival and initial
growth of the explants. Benzyl adenine 5.0ppm was found to be the optimum
cytokinin level for the production of fairly elongated multiple shoots and NAA
0.2ppm was identified as the optimum auxin level for supporting the growth of the
cultures. The normal strength of the inorganic salts and organic growth factors of
the MS medium, with 3 – 4% sucrose or 2 – 3% glucose was found to support the
multiplication and growth of jack shoot cultures. GA3 did not influence the shoot
proliferation or growth. Adenine sulphate at 20ppm was found to increase the
multiplication rate by 27.3%, without affecting the growth of the cultures. Adenine
as well as casein hydrolysate were found to be not beneficial. The Anderson’s
medium was found to be unsuitable to support the proliferation and growth of jack
shoot cultures. Serial subculturing for 10 times at 4 week interval was found to
increase the multiplication rate to 5.39x. The MS medium supplemented with BA
2.0ppm, NAA 0.2ppm and insoluble PVP 500ppm was found to be suitable for shoot
elongation. Half strength of the MS inorganic salts, full strength of the MS organic
factors, 3% sucrose and 0.6% agar were found to be the optimum for the in vitro
rooting of jack shoot cultures. When planted out, the plantlets were observed to
have 55.6% survival.
Callus production was made possible from explants of shoot apices, internodal
segments, leaf segments and root apices. Efforts to induce re-differentiation in the
callus, direct organogenesis and direct/callus mediated somatic embryogenesis were
not successful.
Shoot apices from the seedlings registered a multiplication rate of 17.4x. In this case,
the percentage of rooting was 100, with 6.0 roots formed in 20.75 days. Explants
from fresh stem sprouts of ten year old and thirty year old trees recorded a shoot
multiplication rate of 2.8 and 2.09, respectively in five weeks. In the former, the
rooting percentage was 40 with 2.5 roots produced in 24 days, after 2-3 subcultures.
In the latter, there was 15% rooting with 2.0 roots formed in 46.7 days, after 2-3
subcultures. Explants from six-month old jack grafts failed to produce multiple
shoots and exhibited 50% rooting with 2.0 roots formed in 20.5 days. Cytological
examination revealed the stability of chromosome number in the plantlets.
Anatomical studies revealed the presence of thin culture in the leaves of the young
plantlets.
The procedure for the in vitro clonal propagation of jack through the enhanced
release of axillary buds involved agitating the surface sterilised shoot apices in a
solution of 0.7% insoluble PVP + 2% sucrose for 30-45 minutes and keeping them in
sterile water at 4-50C for 24 hours followed by disinfection (3% sodium
hypochlorite solution for 5 minutes and 0.1% mercuric chloride solution for 10
minutes) and culture in the establishment medium (MS + GA 1.0ppm + activated
charcoal 1.0%) in darkness for four weeks with repeated subculturing. The cultures
were then exposed to light for two weeks, after which the growing shoot apices were
transferred to the proliferation medium (MS + BA 5.0ppm + NAA 0.2ppm +
adenine sulphate 20ppm + insoluble PVP 500ppm). Shoots from the proliferation
medium were transferred after five weeks to an elongation medium (MS + BA
2.0ppm + NAA 0.2ppm + insoluble PVP 500ppm). The shoots were then cultured on
MS medium containing activated charcoal 1.0% for two weeks. For the in vitro root
induction, the shoots were cultured in darkness in 1⁄2 MS + IBA 2.0ppm + NAA
2.0ppm + sucrose 3% + agar 0.6% (for 6 days) and then transferred to 1⁄2 MS
without growth substances for root elongation.
Just after the appearance of the roots, the plantlets were hardened by exposure to
high light intensity (3500 lux) for one week. The plantlets were than transferred to
vermiculture medium, under high relative humidity (90-100%) provided by
microscope covers. The plants were watered with a solution of the MS inorganic
salts at half strength. After another gradual hardening process and as new leaves
were produced, the plantlets were transferred to garden pots and kept in the open
field conditions. The cost of production of one jack plantlet, including one month’s
hardening was worked out to Rs.9.09.
Explants of mussaenda (Mussaenda ervthrophylla Schum. Thonn.) were effectively
surface sterilized by treating with 0.1% mercuric chloride solution for 15 minutes.
The suitable culture establishment medium was identified as MS + BA 0.5ppm +
kinetin 0.5ppm. A shoot multiplication rate of 2.75x was realized in a period of four
weeks on MS medium supplemented with BA 0.5ppm and kinetin 0.5ppm. Sub-
culturing was found to increase the multiplication rate to 2.95x. Full strength of the
MS inorganic salts was found to support the proliferation and growth of mussaenda
shoot cultures. Adenine sulphate, auxins and Anderson’s medium were found to be
not beneficial. The shoots were made to root on MS medium containing half
strength of the inorganic salts and full strength of the organic growth factors, 3%
sucrose, 0.6% agar, 0.4ppm IBA and 0.4ppm NAA, under dark conditions in 37
days. Anderson’s rooting medium was found to be inefficient for the in vitro rooting
of mussaenda shoot cultures. Attempts for planting out were not successful,
probably due to the low number of roots (which were weak and slender), the
development of callus at the root-shoot junction and the partial withering and
yellowing of the shoots by the time the roots were initiated. Attempts for the direct
planting out of the shoots pretreated with IBA solution were not successful.
Segments of ovary wall was identified as the best source of explant for callus
production, registering a callus index value of 400, at the best treatment
(NAA/kinetin combinations 2.0 + 1.0ppm and 4.0 + 2.0ppm). Shoot regeneration
from the callus occurred at a frequency of 33.3% on MS medium supplemented
with BA 2.0ppm or BA/kinetin combinations 0.5 + 0.5ppm and 0.5 + 0.3ppm. Root
regeneration was observed at a frequency of 66.7% with 13.33 roots per culture on
MS medium containing kinetin/NAA combination 2.0 + 8.0ppm after 60 days’
culture. Attempts for direct organogenesis were not successful.
Globular structures resembling somatic embryoids having simultaneous root and
shoot development were observed when the callus from the induction medium (MS
+ 2,4-D 2.0ppm + kinetin 1.0ppm) was transferred to MS medium containing
BA/kinetin combinations 0.5 + 0.5ppm or 1.0 + 0.5ppm after 70 to 73 days culture.
About 4.5 shoots with a tuft of miniature roots were formed per culture, at the best
treatment. Attempts for inducing direct somatic embryogenesis were not successful.
Preliminary studies on culture establishment were made for breadfruit (Artocarpus
altilis L.) and nutmeg (Myristica Fragrans Houtt.). Slight callus production was
made possible in both the cases. Preliminary studies on somatic organogenesis were
made in the case of pepper (Piper nigrum L.). Callus production and
redifferentiation were observed.
Description
PhD
Keywords
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Citation
171034