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High Frequency Microcloning of Aloe Vera and Their True-To-Type Conformity by Molecular Cytogenetic Assessment of Two Years Old Field Growing Regenerated Plants

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High Frequency Microcloning of Aloe Vera and Their True-To-Type Conformity by Molecular Cytogenetic Assessment of Two Years Old Field Growing Regenerated Plants

Sk Moquammel Haque et al. Bot Stud.

Abstract

Background: Aloe vera (L.) Burm.f is an important industrial crop, which has enormous application in pharmaceutical, cosmetic and food industries. Thereby, the demand for quality planting material of A. vera is increasing worldwide. Micropropagation is the widely accepted practical application of plant biotechnology that has gained the status of a multibillion-dollar industry throughout the world and this techniques can be used to meet the industrial demand of A. vera. Present studies aim to develop a proficient methods of high-frequency true-to-type plantlet regeneration without intermediate callus phase for A. vera.

Results: Nodal portion of rhizomatous stem of A. vera were cultured on Murashige and Skoog (MS) medium (Physiol. Plant. 15:473 - 497, 1962) supplemented with various cytokinin and A. vera leaf gel (AvG) as organic supplement. Number of proliferated shoots per explant was increased along with the regeneration cycles and on MS medium supplemented with 2.5 mg/L 6-benzylaminopurine and 10.0% (v/v) AvG, only 17.8 ± 0.35 shoots per explant were induced on 1st regeneration cycle whereas on 3rd regeneration cycle these number increase to 38.5 ± 0.44 shoots per explant on the same medium composition. AvG have an encouraging role to increase the proliferation rate and on 3rd regeneration cycle 27.6 ± 0.53 shoot per explant induced on 2.5 mg/L BAP, but these number increase to 38.5 ± 0.44 shoots per explant when 10.0% (v/v) AvG was added along with 2.5 mg/L BAP. After transfer of individual excised shoots to a one-third strength MS medium containing 20.0% (v/v) AvG, all the shoots formed whole plantlets with maximum number (9.6 ± 0.29) of roots per shoot. 95.0% of the regenerated plantlets survived on poly-green house. Normal flower appeared in 84.2% field growing micropropagated plants after 18 to 20 months of field transfer. Further, clonal fidelity of the two years old micropropagated plants was established by studying mitotic and meiotic chromosomal behavior and also considered the chromosome number and structural organization. There were no alterations in chromosome phenotypes, somatic haploid (pollen mitosis) and diploid chromosome count (n = 7; 2n = 14), or meiotic behavior. Randomly amplified polymorphic DNA analyses revealed there were no somaclonal variations among these regenerants.

Conclusions: These results confirm the very reliable method for large scale production of true-to-type plantlets of A. vera, which can be used for commercial purpose.

Keywords: Aloe vera leaf gel; Diploid and haploid karyotype; Meiotic study; Micropropagation, RAPD fingerprinting; True-to-type regenerants.

Figures

Figure 1
Figure 1
Different stages of Micropropagation and field performance of Aloe vera . (A) Multiple shoots induced in MS medium supplemented with 2.5 mg/L BAP on third regeneration cycle (bar = 1 cm). (B) Multiple shoots induced in MS medium supplemented with 2.5 mg/L BAP and 10.0% AvG on third regeneration cycle (bar = 1 cm). (C) Complete plantlets with root system (bar = 1 cm). (D) Hardening of regenerated plants (bar = 10 cm). (E) Field grown regenerated plants of 18 months old (bar = 10 cm). (F) 22 months old regenerated plant with inflorescence (bar = 10 cm).
Figure 2
Figure 2
Mitotic and Meiotic metaphase plates and karyogram of regenerated Aloe vera plants. (A) Metaphase plate of pollen mitosis showing n = 7 chromosomes. (B) Karyogrm of haploid pollen grain. (C) Mitotic metaphase plate of root-tip cell showing 2n = 14 chromosomes. (D) Karyogram of diploid somatic cell. (E) Metaphase-I of meiosis of pollen mother cell showing 7 pairs of bivalent chromosomes (F) Karyogram of meiotic bivalent chromosomes.
Figure 3
Figure 3
Aceto-carmine stained normal (A-M) & abnormal (N-T) meiotic stages of Aloe vera. (A) Leptotene stage. (B) Zygotene stage showing chromatin threads (C) Pachytene stage showing beaded chromosomes. (D) Diplotene stage showing ‘X’ shaped chiasma. (E) Diakinesis showing 7 bivalents with 4 long and 3 short chromosome pairs. (F) Side view of metaphase-I. (G) Side view of anaphase-I. (H) Side view of telophase-I. (I) Side view of prophase-II (J) Side view of metaphase-II. (K) Side view of anaphase-II. (L) Side view of telophase-II. (M) Tetrad showing 4 pollen grains. (N) Side view of anaphase-I with single chromosomal bridge and lagged chromosome. (O) Side view of anaphase-I with double chromosomal bridge. (P) Side view of anaphase-I with single chromosomal bridge (Q) Telophase-I with lagged chromosome. (R) Metaphase-II with lagged chromosome. (S) Telophase-II with single chromosomal bridge. (T) Telophase-II with univalent laggard chromosome.
Figure 4
Figure 4
RAPD banding profile of both mother plant and field grown micropropagated plants of Aloe vera using OPK-10 primer showing 6 monomorphic bands ranging from 600 bp to 2500 bp (Lane ‘L-1’ = 100 bp plus DNA ladder, Lane ‘M’ = Mother plant, Lane ‘1-10’ = ten different micropropagated plants, Lane ‘L-2’ = 1 kb DNA ladder).

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