مطالعه و بررسی بهینه سازی محیط کشت گل راعی (Hypericum perforatum) با هدف کال زایی و باززایی ریزنمونه های برگ و ساقه و ریشه زایی ساقه

نوع مقاله : مقالات پژوهشی

نویسندگان

1 فردوسی مشهد

2 دانشگاه فردوسی مشهد

چکیده

گل راعی گیاهی چندساله و دارای مواد موثره با ارزشی همچون هایپریسین، سودوهایپریسین و هایپرفورین می باشد که از حیث اثرات ضدافسردگی، ضدسرطانی و افزایش ایمنی بدن اهمیت دارد. کاربرد تکنیک های کشت بافت در این گیاه و هدایت آن به سمت تولید مواد دارویی ارزشمند گیاهی است. لذا این پژوهش با هدف بهینه سازی محیط کشت در راستای تولید کالوس، باززایی و ریشه زایی صورت گرفت. این پژوهش به صورت دو آزمایش مجزا در قالب طرح کاملا تصادفی اجرا گردید. آزمایش اول شامل شش تیمار هورمونی با سطوح متفاوتی از بنزیل آدنین و توفوردی بر روی دو ریزنمونه برگ و ساقه، در دو شرایط نور و تاریکی بود. نمونه های تیمار شده از لحاظ میزان کال زایی و باززایی مورد بررسی قرار گرفتند. در آزمایش دوم نمونه های باززایی شده از ریزنمونه ساقه به منظور ریشه زایی به محیط کشت با چهار تیمار هورمونی شامل بنزیل آدنین و ایندول بوتیریک اسید انتقال یافتند. نتایج نشان داد، در آزمایش اول برای ریزنمونه برگ تیمار بنزیل آدنین با غلظت mg/l 3 و توفوردی با غلظت mg/l5/2 با 98 درصد و برای ریزنمونه ساقه تیمار بنزیل آدنین با غلظت mg/l4 و توفوردی با غلظت mg/l 5/2 با 95 درصد بیشترین میزان کال زایی را داشتند. از نظر باززایی نیز در ریز نمونه های برگ و ساقه به ترتیب تیمار های حاویmg/l 4 و mg/l3 بنزیل آدنین 8 و 62 درصد باززایی نشان دادند. در آزمایش دوم نمونه های باززا شده کالوس ساقه نیز در تیمار حاوی IBA با غلظت mg/l 1 و تیمار فاقد هورمون بیشترین میزان ریشه زایی و در تیمار حاوی mg/l 5/0 بنزیل-آدنین همراه با IBA mg/l 1 و تیمار فاقد IBA به ترتیب 100 و 86 درصد پرآوری نشان دادند و ریشه زایی نداشتند.

عنوان مقاله [English]

Medium Optimization for Callus Induction, Shoot Regeneration and Rooting of Hypericum perforatum from Stem and Leaf Explants

نویسندگان [English]

  • Negin Afsharzadeh 1
  • m azizi 2
  • Leila Samiei 1
1 Ferdowsi University of Mashhad
2 Ferdowsi University of Mashhad
چکیده [English]

Introduction

Hypericum perforatum is a perennial plant that has been used in traditional medicine. H. perforatum have several types of medicinal compounds including antiviral compounds, antioxidants, flavonoides and also has valuable compounds such as Hypericin, Hyperforin, Pseudo-hypericin and xanthones that have effect on human physiology. Aerial parts of H. perforatum are dotted with dark glands that appear as black or red nodules. Black glands were known as localization of secondary metabolites. as a consequence of the commercial potential of this species attempts have been at plant improvement by application of in vitro culture methods. Among seedling explants of H. perforatum, it appears that roots are superior for shoot regeneration. It is generally accepted that explants source is an important factor r successful establishment of tissue culture in many cases. Production of secondary metabolites via plant cell and tissue culture yields various advantage es, including standardization and quality. These criteria are also valid for the main economically important chemical in st. John’s wort, namely hypericin, pseudo hypericin and hyperforin. The aim of this study was to evaluate the effectof some tissue cultures on plant Callogenesis, regeneration and also, study the effect of cytokinin and auxin on rooting rate and shoot multiplication.

Materials and Methods:

This research included two experiments; first experiment plan was a completely randomized in the form of a factorial. Second experiment plan was completely ranom.

first part of experiment : this part were conducted with two explants, leaf and shoot ,maintained in light and dark condition. Shoot explants were derived from sterile seedlings that was obtained from seeds were cultured on MS medium. Seeds were decontaminated by NaClO 20% (V/V) for 20 min and were washed with sterile deionized water. Leaf explants were derived from seedlings in the in vivo condition and decontaminated by NaClO 20% (V/V) for 20 min then washed with sterile deionized water. Both of explants cultured on MS media supplemented with BA(3 and 4 mg/l) and 2,4-D (0, 1 and 2.5 mg/l). Callogenesis and regeneration was measured after 4 weeks.

Second part of experiment: shoot of indirect regeneration for rooting study, were cultured on ½ MS media supplemented with BA (0 and 5/0 mg/l) and IBA (0 and 1 mg/l). Proliferation, shoot and root length were measured after 4 and 8 weeks.

Results and Discussion:

Effects of the factors on first part of experiment; calluses of shoot and leaf explants were induced after 4 weeks. Shoot explants Medium supplemented with 4 mg/l BA and 2.5 mg/l 2, 4-D showed 95% Callogenesis. Leaf explants Medium supplemented with 3 mg/l BA and 2.5 mg/l 2, 4-D showed 98% Callogenesis. Shoot explants Medium supplemented with BA 3 mg/l showed 62% regeneration and leaf explants Medium supplemented with BA 4 mg/l showed 8% regeneration.

For second part of experiments; root induction on half strength medium without hormone and medium supplemented with 0.5 mg/l BA and 1 mg/l IBA had highest rooting frequencies. Average of root length was registered 5.25 cm. half strength medium supplemented with 0.5 mg/l BA had 100% and also, medium with 0.5 mg/l BA and 1 mg/l IBA had 86% shoot multiplication and were not appeared any roots. Average Shoot length on medium without hormone and medium supplemented with 0.5 mg/l BA and 1 mg/l IBA was registered 6.24 cm and in media with 0.5 mg/l BA and 1 mg/l IBA was registered 1.8 cm. based on result of this experiments, the concentration levels of the two hormone BA and 2, 4-D in the induction of calli formation and regeneration of the H. perforatum have been effective. In the second experiment, hormone BA, in the absence of IBA did not cause rooting and increased the degree of shoots and ultimately proliferation was effective. Also, in treatment with 0.5 mg/l BA and 1 mg/l IBA, although the concentration of cytokinin was less than auxin, rooting was not done.

conclusion

The goal of this study was to introduce the suitable medium for Callogenesis and regeneration of H. perforatum for production and breeding aims. Tissue and cell culture methods are used for various purposes. The result of each section can be used effectively in research topics, including corrective tests, processes for increasing secondary metabolites, as well as increasing commercial products. Therefore, by introducing efficient and effective methods, it is possible to increase the production of plants by micropropagation in less time.


مقالات آماده انتشار، پذیرفته شده
انتشار آنلاین از تاریخ 16 فروردین 1400
  • تاریخ دریافت: 07 آذر 1396
  • تاریخ بازنگری: 13 آذر 1400
  • تاریخ پذیرش: 16 فروردین 1399