Screening and kinetic studies of catharanthine and ajmalicine accumulation and their correlation with growth biomass in Catharanthus roseus hairy roots

Figures & data

Table 1. Response of explants of C. roseus cultivars infected with Agrobacterium rhizogenes strains A4 and AR15834 after different co-cultured period.

Cultivars	Bacterial strain	Periods of co-culture: explants-bacteria (days)	Hairy root induction (%)	Number of roots per explants	Average time for the appearance of the first root (days)
	A4	0	78.57	4.5^H	14.23
		2	83.33	5.41^F	12.5
Cooler lilac		3	83.33	10.27^A	11.94
	AR15834	0	55.5	4^I	17.22
		2	89.5	5.6^F	15.46
		3	90.0	8^D	12.55
	A4	0	37.5	1.44^J	16.13
		2	50.0	4.77^G	14.23
Cooler blush		3	58.33	8.29^C	11.82
	AR15834	0	75.0	4.83^G	15.11
		2	95.0	6.08^E	14.5
		3	100	9.55^B	12.03

Letters (A–J) indicate the homogenous groups (Tukey test).

Figure 1. Morphology of induced hairy roots. (A and B) Induction of hairy root on the wounded site of explants after the 30th day of culture in half-strength B5 semi-solid medium; (C, D and E) the selected hairy root lines; (F, G and H) growth of the LP10 hairy roots line after the 14th, 29th and 32nd days of culture in half-strength B5 liquid medium, respectively.

Table 2. Variation of growth index in selected hairy root lines after 20 d of culture in half-strength B5 liquid medium.

Cultivar	Selected Lines	Growth index (GI)^a
Cooler lilac	L2	5.49 ± 1.59
	L12	4.39 ± 0.55
	L14	6.15 ± 0.27
	L17	13.53 ± 1.21
	L54	21.03 ± 1.91^b
Cooler blush	LP10	24.48 ± 1.13 ^b
	LP15	2.41 ± 0.50
	LP16	2.0 ± 1.38
	LP3	2.49 ± 1.13
	LP21	14.10 ± 1.02^b

^a

^bSelected lines according to good growth index.

Figure 2. PCR amplification of rolB gene in hairy root lines of C. roseus. Lane M, molecular weight marker (1000 bp ladder); lane 1, DNA from non-transformed roots; lane 2, DNA from the plasmid A4 (positive control (1); lane 3, DNA from the plasmid 15834 (positive control (2); lane 4, DNA from HR line L54; lane 5, DNA from HR line LP21; lane 6, DNA from HR line LP10.

Figure 3. Dry weight accumulation kinetics in three selected hairy root lines.

Figure 4. Evolution of the catharanthine accumulation in the three selected hairy root lines of C. roseus.

Figure 5. Evolution of ajmalicine accumulation in three selected hairy root lines in C. roseus.

Figure 6. LCMS/MS of the alkaloids of methanolic extract of Catharanthus roseus roots. (A and B) Single ion chromatogram (SIC) of catharanthine [M + H]+parent ion (m/z = 143.50–144.50 + 172.50–173.50) and ajmalicine [M + H]+parent ion (m/z = 143.50–144.50 + 177.50–178.50 + 209.50–210.50 + 221.50–222.50), respectively. (C and D) MS spectrum of catharanthine and ajmalicine, respectively. (E and F) MS² spectrum of catharanthine and ajmalicine, respectively.

Table 3. Relationship between biomass growth and alkaloids accumulation of the three selected lines in C. roseus.

	Coefficient of correlation in alkaloid
DW of hairy root line	Catharanthine	Ajmalicine
LP10	0.4697**	0.5337**
LP21	0.5635**	0.2994*
L54	0.8399***	0.4786**

*, ** and *** significance level at 0.5; 0.1 and 0.1 value of p, respectively.