Research oriented..

1. DEPARTMENT OF LIFE SCIENCES
AKASH VEERSHETTY
Central university of Karnataka
Dept. of life sciences
MSc Plant sciences
Roll no. 2019MLS01

2. Endosperm-derived triploid plant regeneration in diploid Actinidia kolomikta , a cold-hardy kiwifruit relative
Issei Asakuraa , Yoichiro Hoshinoa,b,∗
Abstract :
• Endosperm derived from fruits harvested 6–10 weeks after flowering (WAF) was used for the initial explants to examine the effect of plant growth regulators
on callus formation and organogenesis.
• The endosperm-derived calli induced in MS medium with above 1.0 mg L−1of 2,4-dichlorophenoxyaceticacid (2,4-D) were soft, small, and translucent, and no
morphogenic response was observed.
• The endosperm-derived yellow-green calli induced in the medium with 2,4-D (0.1 mg L−1) plus kinetin(1.0, 2.0, and 5.0 mg L−1) were hard, compact, fast
growing, and showed adventitious shoot primordia.
• These primordia elongated into shoots when the calli were transferred to MS medium supplemented with zeatin (1.0, 2.0, and 4.0 mg L−1) under continuous
light conditions.

3. • The shoots developed roots on half-strength MS medium lacking plant growth regulators.
• In total, 14 plants were obtained. Flow cytometry and chromosomal analysis confirmed that the regenerated plants were triploid, suggesting that
the plant tissue originated from the endosperm.
• This study reveals that the endosperm of A. kolomikta has plant regeneration abilities and that the regenerated plants have the same ploidy level as
the endosperm(triploid).

4. Introduction
• The endosperm formed inside the seeds of most flowering plants as part of the double fertilization process.
• The endosperm cell contains three sets of chromosomes, i.e., it is a triploid (3x) cell.
• The endosperm surrounds the embryo and provides the nutrition necessary for embryo development.
• The genus Actinidia comprises 76 species and approximately 120taxa (Ferguson and Huang, 2007) distributed across a wide geo-graphical range from the
tropics (latitude 0◦) to cold, temperate regions (latitude 50◦N) in Southeast and East Asia
• Kolomikta cultured on MS medium supplemented with 2,4 dichloro phenoxy acetic acid (2,4-D, 2.0 or 4.0 mg L−1) and kinetin (5.0 mg L−1).Highly cold
tolerant and fruits are rich in vitamin- C

5. Results
Effects of developmental stages and preparation of the explants on callus induction

7. Effect of plant growth regulators on callus induction from7-WAF endosperm
• On medium without 2,4-D or supplemented with kinetin only, no callus formation was observed in any of the explants tested .
• Endosperm-derived calli induced on media with more than 1.0 mg L−1of 2,4-D were soft, small, translucent and slow growing (callus types D–L);
• Calli induced on media with 0.1 mg L−12,4-D and 1.0, 2.0, or 5.0 mg L−1kinetin were yellow-green, hard , compact, and fast growing (callus types
A–C)
• The results indicate that callus induction and the resulting callus characteristics were dependent on the concentrations and combinations of 2,4-D
and kinetin.

9. Effect of zeatin on shoot formation and elongation
• To promote shoot elongation and stimulate additional shoot formation in calli, the cytokinin zeatin was included in the v culture medium.
• Calli greater than ca. 5 mm in diameter were transferred to MS medium supplemented with 1.0, 2.0, and 4.0 mg L−1zeatin.
• The cultures were incubated at 22◦C under continuous light conditions (35 mol m−2s−1).
• Twenty-eight cultures were initiated using calli of different types and were cultured in medium containing different concentrations of zeatin (1.0, 2.0, or
4.0 mg L−1).
• One callus each of the A, B, and C types, which had adventitious shoot primordia, showed shoot elongation from the primordia.

11. Rooting of shoots and acclimatization
• To promote root formation and further plant growth, shoots of approximately 5–10 mm length from the above experiments were removed from the calli
and transferred to half-strength MS medium lacking plant growth regulators.
• The cultures were incubated at 22◦C under continuous light (35 mol m−2s−1).
• The plantlets reached approximately 5 cm in height, a process of acclimatization was initiated.
• the shoots originated from A, B, and C types of calli that responded to zeatin .
• All the 14 shoots developed roots after 3–10 weeks of culture and grew into plantlets .

12. Callus induction and
plant regeneration from
cultured endosperm in
Actinidia kolomikta.
(a) A 7-WAF seed
(right), endosperm
(central), and embryo
(left).The endosperm
was used as explant
material in this study.
(b) Type I callus
which is
translucent, soft,
small, and slow
growing. This type
of callus , as well as
callus types D–H
and J–L, showed no.
morphogenic
response
(c) Type B calli
were yellow-green,
hard, compact, and
fast growing. This
type of callus, as
well as type A and
C, developed
adventitious shoot
primordia (inset).
(d) Enlarged view
of adventitious
shoot primordia
(black arrows).

13. (e) Type B callus forming many adventitious shoots after being transferred onto MS
medium with zeatin (2.0 mg L−1).
(f) A rooted plantlet 3 weeks after transplantation onto half-strength MS medium without
plant growth regulators.

14. Confirmation of ploidy level by flow cytometric analysis
• Analysis Flow cytometric analysis was performed to determine ploidy levels in regenerated plantlets.
• Nuclear samples extracted from the leaves were stained with 4,6-diamidino- 2-phenylindole (DAPI), and nuclear fluorescence was measured using a ploidy
analyzer .
• The leaves of diploid A. kolomikta were used as an internal control.
• The DNA content of the sample was estimated by calculating the ratio of relative fluorescence intensity of the sample to that of the internal control.
• We observed fluorescence peaks approximately 1.5 times higher than for diploid leaves of A. kolomikta , confirming the triploid nature of the endosperm-
derived plants .

15. Flow cytometric analysis of ploidy level in endosperm-derived plants. Relative fluorescence
intensities obtained from DAPI-stained nuclei were measured in samples from endosperm-derived
shoots obtained from type A, B, and C calli. The white arrow indicates diploid leaves (Actinidia
kolomikta) as an internal control; the black arrow indicates leaves from endosperm-derived plant.
More than 5000 cells were analyzed for each measurement. The fluorescence peak of the
endosperm-derived plant was almost 1.5 (1.492) times higher than that of the diploid, indicating
that it was triploid.

16. Discussion
• We found that endosperm from 8-WAF fruits provided the highest rate (60%) of callus formation.
• Morphology, texture, and plant regeneration ability of the callus was dependent on the concentration and combination of plant growth regulators, 2,4-D
and kinetin .
• In the present study, soft, small, and translucent calli were induced on MS media with more than 1.0 mg L−1of2,4-D and showed no morphogenic
response.
• we found that yellow-green calli were induced on the medium with 2,4-D (0.1 mg L−1) and kinetin (1.0, 2.0, and 5.0 mg L−1);

17. • In the present study , we tested zeatin for its effect on shoot induction, as it is a widely used cytokinin for adventitious shoot induction in callus cultures.
• In our study, zeatin alone was effective for shoot elongation, although the effect of GA3was not examined. In the present study, flow cytometric analysis
and chromosome observation revealed that the tested shoots were triploid.
• This suggests that the present regeneration system was stable in terms of ploidy variation

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